Methods using a nonlinear optical technique for detection of interactions involving a conformational change

ABSTRACT

A nonlinear optical technique, such as second or third harmonic or sum or difference frequency generation, is used to detect binding interactions, or the degree or extent of binding, that comprise conformational change. In one aspect of the present invention, the nonlinear optical technique detects a conformational change in a probe due to target binding. In another aspect of the invention, the nonlinear optical technique screens candidate probes by detecting a conformational change due to a probe-target interaction. In another aspect of the invention, the nonlinear optical technique screens candidate modulators of a probe-target interaction by detecting a conformational change in the presence of the modulator.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/571,342 filed on Sep. 30, 2009, which is acontinuation of U.S. patent application Ser. No. 11/327,199, filed onJan. 5, 2006, now abandoned, which is a continuation of U.S. patentapplication Ser. No. 10/164,915, filed on Jun. 6, 2002, now abandoned,which claims the benefit of U.S. Provisional Application No. 60/362,003,filed on Mar. 5, 2002; U.S. Provisional Application No. 60/354,679,filed on Feb. 6, 2002; U.S. Provisional Application No. 60/354,668,filed on Feb. 6, 2002; and U.S. Provisional Application No. 60/351,879,filed on Jan. 24, 2002, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method for detecting interactionsbetween biological components using a surface-selective nonlinearoptical technique. In one aspect of the present invention this relatesto detection of binding between probes and targets that results in aconformational change.

BACKGROUND OF THE INVENTION Detection of Binding

Detecting and quantifying interactions such as binding betweenbiomolecules is of central interest in modern molecular biology andmedicine. Ligand and drug binding to molecules is an important theme inmodem biology, medicine and drug development. Binding reactions betweenbiological components often result in a conformational or dipole moment(or induced dipole moment) change. Conformational change (a change inorientation or position of a molecule or subpart(s) of a molecule) is anessential feature of signaling in many systems. A technique fordetecting activation of a signal transducer (e.g., a receptor in amembrane), through a direct measure of conformational change or changein dipole moment, would be of value in basic research and drugdiscovery. For example, high-throughput drug screening for potentialagonists or antagonists of a receptor can be carried out using thepresent invention as a method for detection of whether potentialagonists or antagonists induce a conformational change-indicative ofbinding and activation of a receptor. Because the conformational changeis a direct indicator of receptor activation, it is an excellent meansof screening for drugs; current techniques often rely on indirectmeasures of activation such as changes in fluorescence intensity thatare concomitant with changes in Ca²⁺ ion concentration, which in turn iscaused by the receptor activation. Current techniques that directlymeasure activation, such as patch-clamping techniques with an ionchannel protein, are not amenable to high-throughput scaleup and requirea skilled technician to operate, resulting in higher cost to the user.

Fluorescence-based or surface plasmon-based detection are used to detectbinding interactions, with varying success and efficiency. Problemsusing fluorescence include the presence of a natural fluorescentbackground in many (non-labeled) biological samples as well asphotobleaching. Detection of orientational changes accompanying targetbinding is difficult to do using fluorescence as the technique is notvery sensitive to label orientation: fluorescence polarization, notbeing a coherent technique, is sensitive to rotational motion during thefluorescence lifetime that makes it difficult to assign small measuredchanges in a particular polarization direction to conformational changes(rather than due to rotational motion). It is also difficult to separatesmall changes in probe orientation from the large fluorescent backgroundthat may be present in many biological samples. Furthermore, it is oftennot trivial to separate a signal indicative of binding from oneindicative of a receptor activation using fluorescence; for example,fluorescently tagged targets are tested for their ability to bind to agiven receptor using fluorescence polarization—but binding of a targetto a surface such as a cell surface can occur non-specifically such thatthe target does not specifically bind to the receptor of interest, yetcan still give a signal indicative of said binding; direct detection ofconformational change as an indicator of receptor activation is a moredirect means of assaying for activation. Wavelength-based changes due tochanges in the microenvironment of the label as a result ofconformational change can be followed, but not all conformationalchanges lead to a change in microenvironment and it may be difficult toassign relative changes due to microenvironment to the degree ofconformational change that actually occurs. Examples offluorescent-based prior art to detect conformational changes in proteinsinclude the following: Mannuzzu et al. (1996) Science 271, 213-216describe the direct physical measure of conformational rearrangementunderlying potassium channel gating using an fluorescently-taggedchannel protein. Gether et al., “Fluorescent labeling of purified P2adrenergic receptor,” J. Biol. Chem. 270, 28628-28275 (1995), describethe fluorescent labeling of soluble purified 02 adrenergic receptor todetect ligand-specific conformational changes. Turcatti et al., “Probingthe Structure and Function of the Tachykinin Neurokinin-Receptor throughBiosynthetic incorporation of fluorescent amino acids at specificsites,” J. Biol. Chem. 271, 19991-19998, (1996), describe theincorporation of non-natural fluorescent amino acids into a receptor tomonitor ligand binding. Liu et al., “Site-Directed fluorescent labelingof P-glycoprotein on cysteine residues in the nucleotide bindingdomains,” Biochemistry 35, 11865-11873, (1996), describe fluorescentlabeling of soluble purified P-glycoprotein to detect ligand binding.

Fluorescence has also been used to detect binding of targets to ionchannel receptors when the binding leads to a change in transmembranepotential in cells. The transmembrane potential change (e.g., adepolarization) leads to a change in the intensity, lifetime,wavelength, etc. of the fluorescent or nonlinear-active label in themembrane. Apart from various problems in the detection itself-concerningphotobleaching, artifacts and background noise, these methods onlyprovide indirect assays for the probe-target binding. For example, it ispossible that a given target binds to ion channel probes and the ionchannels are activated, but that this does not lead to a change intransmembrane potential, either for natural reasons or because there-areproblems in the cells in the normal mechanism for producing the changein transmembrane potential. It is desirable therefore to have a direct,optical means of detecting probe-target binding reactions in cases wherethe binding reaction results in a change in orientation or conformationof the probe.

Molecular Beacons

Molecular beacons are also used for the detection of bindinginteractions. A molecular beacon (MB) probe is well known in the art asa hairpin-loop, single-stranded oligonucleotide comprising a probesequence embedded within complementary sequences that form a hairpinstem. The loop portion of the molecule can form a double-stranded DNA inthe presence of complementary nucleic acid. A fluorophore is covalentlyattached to one end of the oligonucleotide, and a nonfluorescentquencher is covalently attached to the other end. There are typicallyfive to eight bases at each side of the two ends of the beacon which arecomplementary to each other. The stem keeps these two moieties in closeproximity to each other, causing the fluorescence of the fluorophore tobe quenched by energy transfer. When the beacon binds to its target, therigidity of the probe-target duplex forces the stem to unwind, whichcauses the separation of the fluorophore and the quencher and therestoration of fluorescence. This permits the detection of probe-targethybrids in the presence of unhybridized probes.

The following (and references therein) describe the production, designand use of molecular beacons (MBs) in the fluorescence literature:

-   Sequence Dependent Rigidity of Single Stranded DNA, Phys. Rev.    Lett., 85, 2400 No. 11, Noel L. Goddard, 1 Gregoire Bonnet, 1 Oleg    Krichevsky, 2 and Albert Libchaber, 2000.-   Metal-containing DNA hairpins as hybridization probes, H. S. Joshi    and Y. Tor, Chem. Commun., 2001, 549-550.-   Spectral Genotyping of Human Alleles, L. G. Kostrikis, S.    Tyagi, M. M. Mhlanga, D. D. Ho, F. R. Kramer, Science v. 279,    February 1998.-   Molecular Beacons for DNA Biosensors with Micrometer to    Submicrometer Dimensions, X. Liu, W. Farmerie, S. Schuster, W. Tan,    Anal. Biochem., 283, 56-63, 2000.-   Multiplex detection of single-nucleotide variations using molecular    beacons, S. A. E. Marras, F. R. Kramer, S. Tyagi, Genetic Analysis:    Biomolecular Engineering, 14 (1999), 151-156.-   Screening unlabeled DNA targets with randomly ordered fiber-optic    gene arrays, F. J. Steemers, J. A. Ferguson, D. R. Walt, Nature    Biotechnology, v. 18, 2000, 91.-   Wavelength-shifting molecular beacons, S. Tyagi, S. A. E.    Marras, F. R. Kramer, Nature Biotechnology, v. 18, 2000, 1191.-   Molecular Beacons: Probes that Fluoresce upon Hybridization, S.    Tyagi, F. R. Kramer, Nature Biotechnology, v. 14, 1996, 303.-   Technology for microarray analysis of gene expression, A. Watson, A.    Mazumder, M. Stewar, S. Balasubramanian, Current Opinion in    Biotechnology, 9, 609-614, 1998.-   Design of a Molecular Beacon DNA Probe with Two Fluorophores, P.    Zhang, T. Beck, W. Tan,-   Angew. Chem. Int. Ed. 40, 402, 2001-   Bonnet G, Tyagi S, Libchaber A, and Kramer F R (1999) Thermodynamic    basis of the enhanced specificity of structured DNA probes. Proc    Natl Acad Sci USA 96, 6171-6176.-   Dubertret B, Calame M, and Libchaber A (2001) Single-mismatch    detection using gold-quenched fluorescent oligonucleotides. Nat    Biotechnol 19, 365-370.-   Marras S A E, Kramer F R, and Tyagi S (1999) Multiplex detection of    single-nucleotide variations using molecular beacons. Genet Anal 14,    151-156.-   Mullah B and Livak K (1999) Efficient automated synthesis of    molecular beacons. Nucleosides & Nucleotides 18, 1311-1312.-   Ortiz E, Estrada G, and Lizardi P M (1998) PNA molecular beacons for    rapid detection of PCR amplicons. Mol Cell Probes 12, 219-226.-   Tyagi S, Marras S A E, and Kramer F R (2000) Wavelength-shifting    molecular beacons. Nat Biotechnol 18, 1191-1196.-   Tyagi S, Bratu D P, and Kramer F R (1998) Multicolor molecular    beacons for allele discrimination. Nat Biotechnol 16, 49-53.-   Tyagi S and Kramer F R (1996) Molecular beacons: probes that    fluoresce upon hybridization. Nat Biotechnol 14, 303-308.-   El-Hajj H H, Marras S A, Tyagi S, Kramer F R, and Alland D (2001)    Detection of rifampin resistance in Mycobacterium tuberculosis in a    single tube with molecular beacons. J Clin Microbiol 39, 4131-4137.-   Giesendorf B A, Vet J A, Tyagi S, Mensink E J, Trijbels F J, and    Blom H J (1998) Molecular beacons: a new approach for semiautomated    mutation analysis. Clin Chem 44, 482-486.-   Kostrikis L G, Tyagi S, Mhlanga M M, Ho D D, and Kramer F R (1998)    Spectral genotyping of human alleles. Science 279, 1228-1229.-   Piatek A S, Telenti A, Murray M R, El-Hajj H, Jacobs W R, Jr.,    Kramer F R, and Alland D (2000) Genotypic analysis of Mycobacterium    tuberculosis in two distinct populations using molecular beacons:    implications for rapid susceptibility testing. Antimicrob Agents    Chemother 44, 103-110.-   Piatek A S, Tyagi S, Pol A C, Telenti A, Miller L P, Kramer F R, and    Alland D (1998) Molecular beacon sequence analysis for detecting    drug resistance in Mycobacterium tuberculosis. Nat Biotechnol 16,    359-363.-   Rhee J T, Piatek A S, Small P M, Harris L M, Chaparro S V, Kramer F    R, and Alland D (1999) Molecular epidemiologic evaluation of    transmissibility and virulence of Mycobacterium tuberculosis. J Clin    Microbiol 37, 1764-1770.-   Szuhai K, Ouweland J M, Dirks R W, Lemaitre M, Truffert J C, Janssen    G M, Tanke H J, Holme E, Maassen J A, and Raap A K (2001)    Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number    quantification in myoclonus epilepsy and ragged-red fibers (MERRF)    syndrome by a multiplex molecular beacon based real-time    fluorescence PCR. Nucleic Acids Res 29, E13.-   Szuhai K, Sandhaus E, Kolkrnan-Uljee S M, Lemaitre M, Truffert J C,    Dirks R W, Tanke H J, Fleuren G J, Schuuring E, and Raap A K (2001)    A novel strategy for human papillomavirus detection and genotyping    with SybrGreen and molecular beacon polymerase chain reaction. Am J    Pathol 159, 1651-1660.-   Tapp I, Malmberg L, Rennel E, Wik M, and Syvanen A C (2000)    Homogeneous scoring of single-nucleotide polymorphisms: comparison    of the 5′-nuclease TaqMan assay and molecular beacon probes.    Biotechniques 28, 732-738.-   Vogelstein B and Kinzler K W (1999) Digital PCR. Proc Natl Acad Sci    USA 96, 9236-9241.-   Arnold S F, Tims E, and McGrath B E (1999) Identification of bone    morphogenetic proteins and their receptors in human breast cancer    cell lines: importance of BMP2. Cytokine 11, 1031-1037.-   Chen W, Martinez G, and Mulchandani A (2000) Molecular beacons: a    real-time polymerase chain reaction assay for detecting Salmonella.    Anal Biochem 280, 166-172.-   Clayton S J, Scott F M, Walker J, Callaghan K, Haque K, Lilogou T,    Xinarianos G, Shawcross S, Ceuppens P, Field J K, and Fox J C (2000)    K-ras point mutation detection in lung cancer: comparison of two    approaches to somatic mutation detection using ARMS allele-specific    amplification. Clin Chem 46, 1929-1938.-   Devor E J (2001) Use of molecular beacons to verify that the serine    hydroxymethyltransferase pseudogene SHMT-psl is unique to the order    primates. Genome Biol 2. 6.1-6.5.-   Dracheva S, Marras S A E, Elhakem A L, Kramer F R, Davis K L, and    Haroutunian V (2001) N-Methyl-D-Aspartic Acid Receptor expression in    Dorsolateral Prefrontal Cortex of elderly patients with    schizophrenia. American Journal of Psychiatry. 158, 1400-1410.-   Durand R, Eslahpazire J, Jafari S, Delabre J F, Marmorat-Khuong A,    di Piazza J P, and Le Bras J (2000) Use of molecular beacons to    detect an antifolate resistance-associated mutation in Plasmodium    falciparum. Antimicrob Agents Chemother 44, 3461-3464.-   Eun A J C and Wong S M (2000) Molecular beacons: a new approach to    plant virus detection. Phytopathology 90, 269-275.-   Fortin N Y, Mulchandani A, and Chen W (2001) Use of real-time    polymerase chain reaction and molecular beacons for the detection of    Escherichia coli O157:H7. Anal Biochem 289, 281-288.-   Gonzalez E, Bamshad M, Sato N, Mummidi S, Dhanda R, Catano G,    Cabrera S, McBride M, Cao X H, Merrill G, O'Connell P, Bowden D W,    Freedman B I, Anderson S A, Walter E A, Evans J S, Stephan K T,    Clark R A, Tyagi S, Ahuja S S, Dolan M J, and Ahuja S K (1999)    Race-specific HIV-1 disease-modifying effects associated with CCR5    haplotypes. Proc Natl Acad Sci USA 96, 12004-12009.-   Heil S G, Lievers K J, Boers G H, Verhoef P, den Heijer M, Trijbels    F J, and Blom H J (2000) Betaine-Homocysteine Methyltransferase    (BHMT): genomic sequencing and relevance to hyperhomocysteinemia and    vascular disease in humans. Mol Genet Metab 71, 511-519.-   Helps C, Reeves N, Tasker S, and Harbour D (2001) Use of real-time    quantitative PCR to detect Chlamydophila felis infection. J Clin    Microbiol 39, 2675-2676.-   Kota R, Holton T A, and Henry R J (1999) Detection of transgenes in    crop plants using molecular beacon assays. Plant Mol Biology Rep 17,    363-370.-   Lewin S R, Vesanen M, Kostrikis L, Hurley A, Duran M, Zhang L, Ho D    D, and Markowitz M (1999) Use of real-time PCR and molecular beacons    to detect virus replication in human immunodeficiency virus type    1-infected individuals on prolonged effective antiretroviral    therapy. J Virol 73, 6099-6103.-   Li Q, Liang J, Luan G, Zhang Y, and Wang K (2000) Molecular    beacon-based homogeneous fluorescence PCR assay for the diagnosis of    infectious diseases. Analytical Sciences 16, 245-248.-   Manganelli R, Dubnau E, Tyagi S, Kramer F R, and Smith I (1999)    Differential expression of 10 sigma factor genes in Mycobacterium    tuberculosis. Mol Microbiol 31, 715-724.-   Martinson J J, Hong L, Karanicolas R, Moore J P, and Kostrikis L    G (2000) Global distribution of the CCR2-641/CCR5-59653T HIV-1    disease-protective haplotype. Aids 14, 483-489.-   McKillip J L and Drake M (2000) Molecular beacon polymerase chain    reaction detection of Escherichia coli O157:H7 in milk. J Food Prot    63, 855-859.-   Metzner K J, Jin X, Lee F V, Gettie A, Bauer D E, Di Mascio M,    Perelson A S, Marx P A, Ho D D, Kostrikis L G, and Connor R I (2000)    Effects of in vivo CD8(+) T cell depletion on virus replication in    rhesus macaques immunized with a live, attenuated simian    immunodeficiency virus vaccine. J Exp Med 191, 1921-1931.-   Park S, Wong M, Marras S A, Cross E W, Kiehn T E, Chaturvedi V,    Tyagi S, and Perlin D S (2000) Rapid identification of Candida    dubliniensis using a species-specific molecular beacon. J Clin    Microbiol 38, 2829-2836.-   Pierce K E, Rice J E, Sanchez J A, Brenner C, and Wangh L J (2000)    Real-time PCR using molecular beacons for accurate detection of the    Y chromosome in single human blastomeres. Mol Hum Reprod 6,    1155-1164.-   Poddar S K (2000) Symmetric vs asymmetric PCR and molecular beacon    probe in the detection of a target gene of adenovirus. Mol Cell    Probes 14, 25-32.-   Poddar S K (1999) Detection of adenovirus using PCR and molecular    beacon. J Virol Methods 82, 19-26.-   Sebti A, Kiehn T E, Perlin D, Chaturvedi V, Wong M, Doney A, Park S,    and Sepkowitz K A (2001) Candida dubliniensis at a Cancer Center.    Clin Infect Dis 32, 1034-1038.-   Shih I M, Zhou W, Goodman S N, Lengauer C, Kinzler K W, and    Vogelstein B (2001) Evidence that genetic instability occurs at an    early stage of colorectal tumorigenesis. Cancer Res 61, 818-822.-   Smit M L, Giesendorf B A, Vet J A, Trijbels F J, and Blom H J (2001)    Semiautomated DNA mutation analysis using a robotic workstation and    molecular beacons. Clin Chem 47, 739-744.-   Steuerwald N, Cohen J, Herrera R J, and Brenner C A (1999) Analysis    of gene expression in single oocytes and embryos by real-time rapid    cycle fluorescence monitored RT-PCR. Mol Hum Reprod 5, 1034-1039.-   Valentin A, Trivedi H, Lu W, Kostrikis L G, and Pavlakis G N (2000)    CXCR4 mediates entry and productive infection of syncytia-inducing    (X4) HIV-1 strains in primary macrophages. Virology 269, 294-304.-   Van Schie R C, Marras S A, Conroy J M, Nowak N J, Catanese J J, and    de Jong P J (2000) Semiautomated clone verification by real-time PCR    using molecular beacons. Biotechniques 29, 1296-1306.-   Vet J A, Majithia A R, Marras S A E, Tyagi S, Dube S, Poiesz B J,    and Kramer F R (1999) Multiplex detection of four pathogenic    retroviruses using molecular beacons. Proc Natl Acad Sci USA 96,    6394-6399.-   Xiao G, Chicas A, Olivier M, Taya Y, Tyagi S, Kramer F R, and    Bargonetti J (2000) A DNA damage signal is required for p53 to    activate gadd45. Cancer Res 60, 1711-1719.-   Zhang L, Lewin S R, Markowitz M, Lin H H, Skulsky E, Karanicolas R,    He Y, Jin X, Tuttleton S, Vesanen M, Spiegel H, Kost R, van Lunzen    J, Stellbrink H J, Wolinsky S, Borkowsky W, Palumbo P, Kostrikis L    G, and Ho D D (1999) Measuring recent thymic emigrants in blood of    normal and HIV-1-infected individuals before and after effective    therapy. J Exp Med 190, 725-732.-   De Baar M P, van Dooren M W, de Rooij E, Bakker M, van Gemen B,    Goudsmit J, and de Ronde A (2001) Single rapid real-time monitored    isothermal RNA amplification assay for quantification of Human    Immunodeficiency Virus Type 1 isolates from groups M, N, and O. J    Clin Microbiol 39, 1378-1384.-   De Baar M P, Timmermans E C, Bakker M, de Rooij E, van Gemen B, and    Goudsmit J (2001) One-tube real-time isothermal amplification assay    to identify and distinguish Human-   Immunodeficiency Virus Type 1 subtypes A, B, and C and circulating    recombinant forms AE and AG. J Clin Microbiol 39, 1895-1902.-   De Ronde A, van Dooren M, van Der Hoek L, Bouwhuis D, de Rooij E,    van Gemen B, de Boer R, and Goudsmit J (2001) Establishment of new    transmissible and drug-sensitive Human Immunodeficiency Virus Type 1    wild types due to transmission of nucleoside analogue-resistant    virus. J Virol 75, 595-602.-   Greijer A. E., Adriaanse H. M., Dekkers C. A., and    Middeldorp J. M. (2002) Multiplex real-time NASBA for monitoring    expression dynamics of human cytomegalovirus encoded IE1 and pp 67    RNA. J Clin Virol 24, 57-66.-   Klerks M M, Leone G 0, Verbeek M, van den Heuvel J F, and Schoen C    D (2001) Development of a multiplex AmpliDet RNA for the    simultaneous detection of Potato leafroll virus and Potato virus Y    in potato tubers. J Virol Methods 93, 115-125.-   Leone G, van Schijndel H, van Gemen B, Kramer F R, and Schoen C    D (1998) Molecular beacon probes combined with amplification by    NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids    Res 26, 2150-2155.-   Lanciotti R. S, and Kerst A. J. (2001) Nucleic acid sequence-based    amplification assays for rapid detection of West Nile and St. Louis    encephalitis viruses. J Clin Microbiol 39, 4506-4513.-   Szemes M., Klerks M. M., van den Heuvel J. F., and    Schoen C. D. (2002) Development of a multiplex AmpliDet RNA assay    for simultaneous detection and typing of potato virus Y isolates. J    Virol Methods 100, 83-96.-   Van Beuningen R, Marras S A E, Kramer F R, Oosterlaken T, Weusten J,    Borst G, and van de Wiel P (2001) Development of a high-throughput    detection system for HIV-1 using real-time NASBA based on molecular    beacons. Proceedings-SPIE the International Society for Optical    Engineering. 4264, 66-71.-   Yates S, Penning M, Goudsmit J, Frantzen I, van De Weijer B, van    Strijp D, and van Gemen B (2001) Quantitative detection of Hepatitis    B Virus DNA by real-time nucleic acid sequence-based amplification    with molecular beacon detection. J Clin Microbiol 39, 3656-3665.-   Brown L J, Cummins J, Hamilton A, and Brown T (2000) Molecular    beacons attached to glass beads fluoresce upon hybridisation to    target DNA. Chemical Comm, 621-622.-   Liu X, Farmerie W, Schuster S, and Tan W (2000) Molecular beacons    for DNA biosensors with micrometer to submicrometer dimensions. Anal    Biochem 283, 56-63.-   Liu X and Tan W (1999) A fiber-optic evanescent wave DNA biosensor    based on novel molecular beacons. Anal Chem 71, 5054-5059.-   Steemers F J, Ferguson J A, and Walt D R (2000) Screening unlabeled    DNA targets with randomly ordered fiber-optic gene arrays. Nat    Biotechnol 18, 91-94.-   Antony T, Thomas T, Sigal L H, Shirahata A, and Thomas T J (2001) A    molecular beacon strategy for the thermodynamic characterization of    triplex DNA: triplex formation at the promoter region of cyclin D1.    Biochemistry 40, 9387-9395.-   Bonnet G, Krichevsky 0, and Libchaber A (1998) Kinetics of    conformational fluctuations in DNA hairpin-loops. Proc Natl Acad Sci    USA 95, 8602-8606.-   Fang X, Li J J, and Tan W (2000) Using molecular beacons to probe    molecular interactions between lactate dehydrogenase and    single-stranded DNA. Anal Chem 72, 3280-3285.-   Gao W, Tyagi S, Kramer F R, and Goldman E (1997) Messenger RNA    release from ribosomes during 5′-translational blockage by    consecutive low-usage arginine but not leucine codons in Escherichia    coli. Mol Microbiol 25, 707-716.-   Goddard N L, Bonnet G, Krichevsky O, and Libchaber A (2000) Sequence    dependent rigidity of single stranded DNA. Phys Rev Lett 85,    2400-2403.-   Gold B, Rodriguez M, Marras S A E, Pentecost M, and Smith I (2001)    The Mycobacterium tuberculosis IdeR is a dual functional regulator    that controls transcription of genes involved in iron acquisition,    iron storage, and survival in macrophages. Molecular Microbiology    42, 851-866.-   Jordens J Z, Lanham S, Pickett M A, Amarasekara S, Abeywickrema I,    and Watt P J (2000) Amplification with molecular beacon primers and    reverse line blotting for the detection and typing of human    papillomaviruses. J Virol Methods 89, 29-37.-   Joshi H S and Tor Y (2001) Metal-containing DNA hairpins as    hybridization probes. Chemical Comm 6, 559-550.-   Kaboev O K, Luchkina L A, Tret'iakov A N, and Bahrmand A R (2000)    PCR hot start using primers with the structure of molecular beacons    (hairpin-like structure). Nucleic Acids Res 28, E94.-   Li J J, Geyer R, and Tan W (2000) Using molecular beacons as a    sensitive fluorescence assay for enzymatic cleavage of    single-stranded DNA. Nucleic Acids Res 28, E52.-   Li J J, Fang X H, Schuster S M, and Tan W H (2000) Molecular    beacons: a novel approach to detect protein-DNA interactions. Angew    Chem Int Ed 39, 1049-1052.-   Lindquist J N, Kauschke S G, Stefanovic B, Burchardt E R, and    Brenner D A (2000) Characterization of the interaction between    alphaCP(2) and the 3′-untranslated region of collagen alphal (I)    mRNA. Nucleic Acids Res 28, 4306-4316.-   Liu J., Feldman P., and Chung T. D. (2002) Real-time monitoring in    vitro transcription using molecular beacons. Anal Biochem 300,    40-45.-   Matsuo T (1998) In situ visualization of mRNA for basic fibroblast    growth factor in living cells. Biochimica Biophysica Acta 1379,    178-184.-   Nazarenko I A, Bhatnagar S K, and Hohman R J (1997) A closed tube    format for amplification and detection of DNA based on energy    transfer. Nucleic Acids Res 25, 2516-2521.-   Saha B K, Tian B, and Bucy R P (2001) Quantitation of HIV-1 by    real-time PCR with a unique fluorogenic probe. J Virol Methods 93,    33-42.-   Schofield P, Pell A N, and Krause D 0 (1997) Molecular beacons:    trial of a fluorescence-based solution hybridization technique for    ecological studies with ruminal bacteria. Appl Environ Microbiol 63,    1143-1147.-   Sokol D L, Zhang X, Lu P, and Gewirtz A M (1998) Real time detection    of DNA.RNA hybridization in living cells. Proc Natl Acad Sci USA 95,    11538-11543.-   Strouse R J, Hakki F Z, Wang S C, DeFusco A W, Garrett J L, and    Schenerman M A (2000) Using molecular beacons to quantify low levels    of type I endonuclease activity. Biopharm 13, 40-47.-   Tan W, Fang X, Li J, and Liu X (2000) Molecular beacons: a novel DNA    probe for nucleic acid and protein studies. Chemistry 6, 1107-1    1111.-   Thelwell N, Millington S, Solinas A, Booth J, and Brown T (2000)    Mode of action and application of scorpion primers to mutation    detection. Nucleic Acids Res 28, 3752-3761.-   Tung C H, Mahmood U, Bredow S, and Weissleder R (2000) In vivo    imaging of proteolytic enzyme activity using a novel molecular    reporter. Cancer Res 60, 4953-4958.-   Whitcombe D, Theaker J, Guy S P, Brown T, and Little S (1999)    Detection of PCR products using self-probing amplicons and    fluorescence. Nat Biotechnol 17, 804-807.-   Yamamoto R, Baba T, and Kumar P K (2000) Molecular beacon aptamer    fluoresces in the presence of Tat protein of HIV-1. Genes Cells 5,    389-396.-   Ying L, Wallace M I, and Klenerman D (2001) Two-state model of    conformational fluctuation in a DNA hairpin-loop. Chemical Physics    Letters 334, 145-150.-   Zhang P, Beck T, and Tan W (2001) Design of a molecular beacon DNA    probe with two fluorophores. Angew. Chem. Int. Ed. 40, 402-405.

Nonlinear Optical Techniques

Surface-selective nonlinear optical techniques have previously beenconfined mainly to physics and chemistry since relatively few biologicalsamples are intrinsically non-linearly active. Examples include the useof an optically nonlinear-active dye as a membrane stain—and anendogenous nonlinear-active stain (GFP) that is used to image biologicalcells (Campagnola et al., “High-resolution nonlinear optical imaging oflive cells by second harmonic generation,” Biophysical Journal 77 (6),3341-3349 (1999), Peleg et al., “Nonlinear optical measurement ofmembrane potential around single molecules at selected cellular sites,”Proc. Natl. Acad. Sci. V. 96, (1999), 6700-6704 and references therein).The following references (and references therein) are exemplary of thisart:

-   A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N.    Friedman, O. Bouevitch, Z. Rothman, L. Loew and M. Sheves, “Second    Harmonic Generation of Biological Interfaces: Probing the Membrane    Protein Bacteriorhodopsin and Imaging Membrane Potential Around GFP    Molecules at Specific Sites in Neuronal Cells of C. elegans,”    Chemical Physics 245, 133 (1999).-   O. Bouevich, A. Lewis, I. Pinnevsky and L. Loew, “Probing Membrane    Potential with Non-linear Optics,” Biophys. J. 65, 672 (1993).-   I. Ben-Oren, G. Peleg, A. Lewis, B. Minke and L. Loew, “Infrared    nonlinear optical measurements of membrane potential in    photoreceptor cells,” Biophys. J. 71, 616 (1996).-   G. Peleg, A. Lewis, M. Linial and L. M. Loew, “Non-linear Optical    Measurement of Membrane Potential Around Single Molecules at    Selected Cellular Sites,” Proc. Acad. Sci. USA 96, 6700 (1999).-   P. Campagnola, Mei-de Wei, A. Lewis and L. Loew, “High-Resolution    Nonlinear Optical Imaging of Live Cells by Second Harmonic    Generation,” Biophys. J. 77, 3341 (1999).-   J. Y. Huang, A. Lewis and L. Loew, “N[on-linear Optical Properties    of Potential Sensitive Styryl Dyes”, Biophysical J. 53, 665 (1988).-   A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N.    Friedman, O. Bouevitch, Z. Rothman, L. Loew and M. Sheves, “Second    Harmonic Generation of Biological Interfaces: Probing Membrane    Proteins and Imaging Membrane Potential Around GFP Molecules at    Specific Sites in Neuronal Cells of C. elegans,” Chemical Physics    245, 133-144 (1999).-   A. Khatchatouriants, A. Lewis, Z. Rothman, L. Loew and M. Treinin,    “GFP is a Selective Non-Linear Optical Sensor of    Electrophysiological Processes in C. elegans,” Biophys. J. (in    press, 2000)

In the prior art, nonlinear-active stains are immobilized in membranesand these stains are used to image the cell surfaces. However, thestains intercalate into the membranes in either an ‘up’ or ‘down’direction, thus reducing the total nonlinear signal due to destructiveinterference. Nonlinear optically active dyes have also been used tomeasure the kinetics of those dyes crossing lipid bilayers in liposomes(A. Srivastava and K B Eisenthal, “Kinetics of molecular transportacross a liposome bilayer,” Chem. Phys. Lett. 292 (3): 345-351 (1998)).

The present invention provides advantages over fluorescence-baseddetection, such as reduced background, reduced photobleaching,simplified optical detection, and no need for labor- or time-consumingwashing steps. These advantages are due to the low background of thenonlinear optical techniques and the fact that the method is ascattering rather than an absorption process.

It is therefore an object of the present invention to provide a direct,optical means of detecting probe-target binding reactions in cases wherethe binding reaction results in a change in orientation or conformationof the probe, target, or both probe and target.

SUMMARY OF THE INVENTION

The invention discloses a method for screening one or more candidatebinding partners (referred to herein as probes) for binding to a testmolecule (referred to herein as targets). The method involvesilluminating a sample with one or more light beams at one or morefundamental frequencies, said sample comprising the test moleculeexposed to the one or more candidate binding partners, and measuring oneor more physical properties of a nonlinear optical light beam emanatingfrom the sample. A change in the value of the one or more physicalproperties measured relative to a value for the one or more physicalproperties measured in the absence of exposure of said test molecule tosaid one or more candidate binding partners indicates that said one ormore candidate binding partners bind said test molecule.

The invention discloses a method for screening one or more candidatemodulator molecules for the ability to modulate an interaction between atest molecule and its binding partner. The method involves illuminatinga sample with one or more light beams at one or more fundamentalfrequencies, said sample comprising said test molecule exposed to (i)said binding partner, and (ii) said one or more candidate modulatormolecules, and measuring one or more physical properties of a nonlinearoptical light beam emanating from said sample. A change in the value ofsaid one or more physical properties measured relative to the value forsaid one or more physical properties measured in the absence of exposureto said one or more candidate modulator molecules indicates that saidone or more candidate modulator molecules modulate the interactionbetween said test molecule and its binding partner.

The invention provides a method for detecting a conformational change ina test molecule upon binding of the test molecule to a binding partnercomprising contacting said test molecule with one or more candidatebinding partners, where the test molecule or the one or more candidatebinding partners is labeled with a nonlinear-active moiety that is notnative to the test molecule or the one or more candidate bindingpartners, respectively. The method involves illuminating said contactedtest molecule with one or more light beams at one or more fundamentalfrequencies, and measuring one or more physical properties of anonlinear optical light beam emanating from said sample. A change in thevalue of said one or more physical properties measured relative to thevalue for said one or more physical properties measured in the absenceof said one or more candidate binding partners indicates that at leastone of said one or more candidate binding partners bind to said testmolecule and that said binding induces a conformational change in saidcandidate binding partners, in said test molecule, or in both saidcandidate binding partners and said test molecule.

The invention provides a method for detecting the degree or extent ofthe binding interaction between a test molecule and one or more bindingpartner comprising contacting said test molecule with one or morecandidate binding partners, wherein the test molecule or the one or morecandidate binding partners is labeled with a nonlinear-active moietythat is not native to the test molecule or the one or more candidatebinding partners, respectively. The method involves illuminating saidcontacted test molecule with one or more light beams at one or morefundamental frequencies, and measuring one or more physical propertiesof a nonlinear optical light beam emanating from the sample. A change inthe value of said one or more physical properties measured relative tothe value for the one or more physical properties measured in theabsence of said one or more candidate binding partners indicates that atleast one of said one or more candidate binding partners binds to saidtest molecule, the degree or extent of the conformational change thatsaid binding induces.

In a specific embodiment, the present invention relates to a method fordetecting interactions between biological components using asurface-selective nonlinear optical technique. In one aspect of thepresent invention this relates to detection of binding between probesand targets that results in a conformational change. The inventiondiscloses methods for screening one or more probes through theconformational changes they induce on binding targets. The inventionalso discloses methods for screening modulators of the probe-targetbinding interaction, and for determining the degree or extent of bindingthrough the conformational changes the binding induces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one embodiment of the apparatus in which the mode ofgeneration and collection of the second harmonic light is by reflectionoff the substrate with surface-attached probes.

FIG. 2 depicts one embodiment of an apparatus in which the mode ofgeneration and collection of the second harmonic light is by totalinternal reflection through a prism. The prism is coupled by anindex-matching material to a substrate with surface-attached probes.

FIG. 3 depicts one embodiment of an apparatus in which the mode ofgeneration and collection of the second harmonic light is by totalinternal reflection through a wave-guide with multiple reflections asdenoted by the dashed line inside the wave-guide.

FIGS. 4A-D depict one embodiment of a flow-cell for delivery and removalof biological components and other fluids to the substrate containingattached probes.

FIGS. 5A-C depict three embodiments of an apparatus in which the mode ofgeneration and collection of the second harmonic light is bytransmission through a sample. In FIG. 5A, the second harmonic beam isco-linear with the fundamental. In FIG. 5B, the second harmonic iscollected from a direction orthogonal to the fundamental (‘right-anglecollection’). In FIG. 5C, the second harmonic light is collected by anintegrating sphere and a fiber optic line.

FIG. 6 depicts an embodiment of the transformation, using a series ofoptical components, of a collimated beam of the fundamental light into aline shape suitable for scanning a substrate.

FIGS. 7A-B depict an embodiments patterned in an array format. FIG. 7Adepicts an embodiment of a substrate surface (containing attachedprobes) which has been patterned into an array format (elements 1-35).FIG. 7B depicts one element of a substrate array in which each elementis a well with walls, with surface-attached probes, and the well iscapable of holding some liquid and serves to physically separate thewell's contents from adjacent wells or other parts of the substrate.

FIG. 8 depicts one embodiment of a surface chemistry used to attacholigonucleotide or polynucleotide samples to the substrate surface.

FIGS. 9A-B depict an embodiment of a substrate containing multiplewells. FIG. 9A depicts the substrate containing multiple wells (1-16),each of which contains surface-attached probes as depicted in FIG. 9B.

FIG. 10 depicts an embodiment of the apparatus substrate with the use ofan aminosilane surface-attached layer on top of a reflective coating.The reflective coating underneath the aminosilane layer improvescollection of the nonlinear optical light. The aminosilane layer issuitable for coupling biomolecules or other probe components to thesubstrate.

FIGS. 11A-B depict an embodiment of an apparatus in which the mode ofgeneration and collection of the second harmonic light is through afiber optic. FIG. 11A depicts the use of a bundle of fiber optic linesand FIG. 11B depicts the use of beads coupled to the end of a fiber forattaching probes.

FIGS. 12A-C depict three embodiments of an apparatus in which the modeof generation and collection of the second harmonic light is bytransmission through a sample. FIG. 12A depicts both the fundamental andsecond harmonic beams travelling co-linearly through a sample. FIG. 122Bdepicts the fundamental and second harmonic beams being refracted at thetop surface (top surface contains attached probes) of a substrate withthis surface generating the second harmonic light. FIG. 12C depicts asimilar apparatus to FIG. 12B except that the bottom surface (bottomsurface contains attached probes) generates the second harmonic light.

FIGS. 13A-B depict two embodiments of an apparatus in which secondharmonic light is generated by total internal reflection at aninterface. The points of generation of the second harmonic light aredenoted by the circles. In FIG. 13A, a dove prism is used to guide thelight to a surface capable of generating the second harmonic light(bottom surface of prism but can also be another surface coupled to theprism through an index-matching material). In FIG. 133B, a wave-guidestructure is used to produce multiple points of second harmonicgeneration.

FIGS. 14A-C depict three embodiments of an apparatus in which secondharmonic light is generated using a fiber optic line (with attachedprobes at the end of the fiber). FIG. 14A depicts an apparatus in whichboth generation and collection of the second harmonic light occur in thesame fiber. FIG. 14B depicts the use of a bead containingsurface-attached probes at the end of the fiber. FIG. 14C depicts anapparatus in which the second harmonic light is generated at the end ofthe fiber optic (containing attached probges) and collected using amirror or lens external to the fiber optic.

FIGS. 15A-B depict two embodiments of an apparatus using an opticalcavity for power build-up of the fundamental.

FIGS. 16A-C depict three embodiments of an apparatus in which the modeof generation and collection of the second harmonic light usesreflection of the light from an interface.

FIG. 17 depicts one embodiment of the sample cell with an appliedelectric field. The sample cell (5) is open and is depicted side-on andfilled with solution (10) containing probes and targets. The fundamentalbeam enters the sample cell to the right (15) and passes throughtransparent electrodes (20) separated by a spacer of 1-3 mm (25). Theelectrodes are connected to a source of voltage (30). When voltage isapplied, the targets and probes become partially oriented, thusproducing the nonlinear optical light (e.g., second harmonic light).

FIG. 18 depicts an apparatus for measuring probe-target interactions insuspended cells. A source of fundamental light from a Ti:Sapphirefemtosecond laser (1000) operating at 800 nm is directed through afilter (RG-645, CVI Laser) that blocks 400 nm light (second harmonic).The fundamental is then focused using plano-convex lens 1200 (100 mmfocal length, Melles Griot) into sample holder 1300 containing asuspension of biological cells. The second harmonic light is collectedparallel to the fundamental direction using a 80 mm diameterplano-convex collimating lens (1400) (120 mm focal length, Melles Griot)that is positioned to have its focus at the focus of lens 1200. Thelight is sent through a color filter (BG-39, CVI Laser) to block thefundamental light and pass second harmonic light on to a secondplano-convex lens 1600 (120 mm focal length) Melles Griot which focusesthe light through a monochromator slit and onto its grating (1700). Aphotomultiplier is attached to the monochromator and detects the secondharmonic light. The signal from the photomultiplier is sent on to photoncounting electronics (SR400, Stanford Research Systems) and then passedto a computer 1900 for storage and analysis of the data.

FIGS. 19A-C depict various embodiments of the present invention. In 19A,receptors embedded in a membrane surface are exposed to ligands orparticles for which they have an affinity. After the binding interactionoccurs, the ligands or particles are bound to the membrane-associatedreceptors. In 19B, cells are attached (or ‘plated’) to a surface orsubstrate to form approximately a monolayer of cells (e.g., ˜100%confluent cells); cells can also be stacked to form multilayers at asubstrate or surface. In both cases in 19B, incident fundamental lightcan be transmitted through the substrate or surface, run parallel to thesurface and through the multilayer or monolayer of cells, coupled to thecell layer evanescently, or some combination thereof FIG. 19C depicts aconformational change process. Receptors in a membrane surface (e.g., ahost cell) are labeled with nonlinear-active labels which, on average,possess an orientation with respect to the surface plane, specificallyan angle of .theta. of the hyperpolarizability with respect to thesurface plane; binding and subsequent activation of the receptor byligands causes the labels to shift orientation to angle 74. Small shiftsin angle can cause a substantial change in a physical property of themeasured nonlinear optical light (e.g., intensity of the light).

FIGS. 20A-B depict a molecular beacon that has been modified to form anonlinear-active analogue. In FIG. 20A, a single strand of nucleotidesis coupled to a nonlinear-active dye (gray-hatch) and an enhancer (opencircle). When the molecular beacon analogue is hybridized to acomplementary target, the dye and enhancer are separated by theconformational change, leading to a measurable change in the nonlinearresponse of the dye. The amount of the change can be made quantitativewith the amount of probe-target hybridization. In FIG. 20B, an exampleof a particular oligonucleotide sequence with attached dye and enhancer(Au particle) is shown (SEQ ID NO: 5). The sequences of various targets(SEQ ID NOs: 1-4, respectively) used for testing the degree ofhybridization to this probe are also shown.

DESCRIPTION OF THE INVENTION

The present invention uses a nonlinear optical technique to detectprobe-target interactions involving a conformational change. Examples ofnonlinear optical technique include but are not limited tosecond-harmonic generation, sum-frequency generation,difference-frequency generation, third-harmonic generation andhyper-Rayleigh scattering HRS). The present invention can be used withany of the nonlinear optical techniques, however many embodimentsdescribe the use of the second-order techniques (e.g., second-harmonicgeneration and hyper-Rayleigh scattering). The following references, andreferences therein, describe the field of nonlinear optics:

-   Nonlinear Optics, R. W. Boyd, 1991, Academic Press.-   The Elements of Nonlinear Optics, P. N. Butcher and D. Cotter,    Cambridge University Press, 1991.

Nonlinear Optical Techniques

Nonlinear optical light is any light that results from a nonlineartransformation of light beams at one or more fundamental frequencies(also referred to herein as fundamental beam(s)). A nonlinear opticaltechnique is capable of transforming the physical properties, such asfrequency, intensity, etc., of one or more incident light beams, calledthe fundamental beams. The nonlinear beams emanating from the sample arethe higher order frequency beams, e.g. second or third harmonic, etc.,or the beams at the sum or difference frequencies. For example, insecond harmonic generation (SHG), two photons of the fundamental beamare virtually scattered by the sample to produce one photon of thesecond harmonic. A nonlinear optical technique is also referred toherein as a surface-selective nonlinear optical technique.

Second harmonic generation (SHG) and other surface-selective nonlinearoptical techniques are directly related to the orientation of thenonlinear-active species in a sample, because the fundamental andnonlinear beams have well-defined phase relationships, and thewavefronts of the nonlinear beam in a macroscopic sample (within thecoherence length) are in phase. Any change in the orientation of thenonlinear-active species can be detected by measuring one or morephysical properties of the nonlinear optical beam emanating from thesample. These coherency properties of the nonlinear optical techniqueoffer a number of advantages useful for surface or high-throughputstudies in which, for example, either a single surface or a microarraysurface is examined. The coherent nature of the nonlinear optical beamemanating from the sample also allows discrimination among more than onenonlinear optical beam emanating from a sample. In alternate embodimentsof the invention, assays can be conducted where multiple fundamentallight beams at one or more frequencies, incident with one or morepolarization directions relative to the sample, can be used, with theresulting emanation of at least two nonlinear beams. An apparatus usingnonlinear optical surface-selective-based detection, such as with secondharmonic generation, requires minimal collection optics since generationof the nonlinear light only occurs at the interface (in the absence ofan applied field) and thus, in principle, allows extremely high depthdiscrimination and fast scanning.

A common equation used to model orientation dependence ofnonlinear-active species at an interface is:

χ⁽²⁾ =N _(s)<α⁽²⁾>

where ω⁽²⁾ is the nonlinear susceptiblity, N_(s) is the total number ofmolecules per unit area at the interface and <α⁽²⁾> is the average overthe orientational distribution of the nonlinearhyperpolarizabilities—α⁽²⁾—in these molecules. Typical equationsdescribing the nonlinear interaction for second harmonic generation are:α⁽²⁾(2ω)=β:E(w)·E(ω) or P⁽²⁾(2ω)=χ⁽²⁾:E(ω)E(ω) where α and P are,respectively, the induced molecular and macroscopic dipoles oscillatingat frequency 2ω, β and χ⁽²⁾ are, respectively, the hyperpolarizabilityand second-harmonic (nonlinear) susceptibility tensors, and E(ω) is theelectric field component of the incident radiation oscillating atfrequency ω. The macroscopic nonlinear susceptibility χ⁽²⁾ is related byan orientational average of the microscopic hyperpolarizability. Thenext order term in the expansion of the induced macroscopic dipoledescribes other nonlinear phenomenon, such as third harmonic generation.The third order term is responsible for such nonlinear phenomena astwo-photon fluorescence. For sum or difference frequency generation, thedriving electric fields (fundamentals) oscillate at differentfrequencies (i.e., ω₁ and ω₂) and the nonlinear radiation oscillates atthe sum or difference frequency (ω₁±ω₂).

The intensity of SHG is proportional to the square of the nonlinearsusceptibility and is dependent on the amount of orientednonlinear-active species in a sample, and thus to changes in thisorientation, both at an interface and species aligned in the bulk (thelatter through an electric field-poled mechanism, for example). Thisproperty can be exploited to detect a conformational change. Forexample, conformational change in receptors can be detected using anonlinear-active label or moiety wherein the label is attached to orassociated with the receptor; a conformational change leads to a changein the direction (orientation) of the label with respect to the surfaceplane (or applied field direction) and thus to a change in a physicalproperty of the nonlinear optical signal. The techniques areintrinsically sensitive to these changes at an interface and can be madesensitive to them in the bulk as well, by applying an electric field topole molecules or simply by detecting that fraction of the ensemblewhich produce hyper-Rayleigh scattering (HRS) due to fluctuationalchanges in their number density or orientation as is well known to oneskilled in the art.

In hyper-Rayleigh scattering (HRS), the fluctuations of nonlinear-activemolecules lead to instantaneous departures from centrosymmetry, and thusallow for a low amount of second-harmonic emission to occur, althoughthis emission is incoherent. Because the. fluctuations depend onmolecular size, among other properties, HRS can be used to discriminatean unbound molecule in solution from the same molecule bound to one ormore binding partners (also referred to herein as probes). Thermalenergy drives the fluctuations required for HRS, however, an externalforce can also be applied to induce or amplify the fluctuations, thusincreasing the HRS signal. For example, a flow-field can be used totransiently orient molecules in solution by injecting a burst or streamof fluid into it. Pulsed and alternating electric fields applied to thesample can also increase the HRS signal.

There are a number of examples in the literature of the use of HRS tomeasure beta (hyperpolarizability) of nonlinear optical molecules. Thepresent invention extends the use of HRS to detect binding interactionsand to screen for test molecules (also referred to herein as targets) ormodulators (described below) capable of binding or modulatingprobe-target interactions.

The following references, and references therein, describe the HRStechnique:

-   Clays, K. et al., “Nonlinear Optical Properties of Proteins Measured    by Hyper-Rayleigh Scattering in Solution”, Science, v. 262 (5138),    1419-22-   Vance, F W, Lemon B. I., Hupp, J. T. Enormous Hyper-Rayleigh    Scattering from Nanocrystalline Gold Particle Suspensions”, J. Phys.    Chem. B 102:10091-93 (1999).

Electric field induced second harmonic (EFISH) is technique well knownin the field of nonlinear optics that can be used according to theinvention to render a system nonlinear-active that is not normally so,such as a bulk phase, through the application of an electric field thatbreaks the symmetry of the bulk phase. In a specific embodiment, theEFISH technique can be used to measure the hyperpolarizability ofmolecules in solution by using a dc field to induce alignment in themedium, and allowing nonlinear-activity (such as SHG) to be observed.This is sometimes called the reorientational mechanism.

EFISH is a third order nonlinear optical effect, with the polarizationsource written as:

P ⁽²⁾(ω₃)=χ⁽²⁾(ω₃;ω₁,ω₂):E ^(ω1) E ^(ω2)

The polarization is the result of the application of two optical fieldsand a static (dc) field. The following references describe applying theEFISH technique to, e.g., liquid and condensed phase samples:

-   R. Dworczak and D. Kieslinger, Phys. Chem. Chem. Phys., 2000, 2,    5057-5064.-   J. L. Oudar, J. Chem. Phys., 1977, 67, 446.-   B. F. Levine and C. G. Bethea, J. Chem. Phys., 1974, 60,-3856.-   B. F. Levine and C. G. Bethea, J. Chem. Phys., 1975, 63, 2666.-   K. D. Singer and A. F. Garito, J. Chem. Phys., 1981, 75, 3572.-   C. G. Bethea, Applied Optics, 1975, 14, 1447.-   B. F. Levine and C. G. Bethea, J. Chem. Phys., 1976, 65, 1989.-   B. F. Levine and C. G. Bethea, J. Chem. Phys., 1974, 60, 3856.-   B. F. Levine, J. Chem. Phys., 1975, 63, 115.-   B. F. Levine and C. G. Bethea, J. Chem. Phys. 1977, 66, 1070.-   J. L. Oudar and D. S. Chemla, J. Chem. Phys., 1977, 66, 2664.-   R. S. Finn and J. F. Ward, J. Chem. Phys., 1974, 60, 454.-   B. F. Levine and C. G. Bethea, Appl. Phys. Lett., 1974, 24, 445.

In addition, the electrodes used to apply the electric field can bespatially patterned (periodically patterned) to achieve quasi-phasematching. Quasi-phase matching is a well known technique for increasingthe effective nonlinear path length in a nonlinear-active material, byalternating the nonlinear susceptibility of a material with a period ofa coherence length. The following references (and references therein)describe this technique:

-   M. Jager et al., Appl. Phys. Lett. 68, 1996, 1183.-   M. M. Fejer et al., IEEE Journal of Quantum Electronics, v. 28,    1992, 2631.-   G. D. Landry et T. A. Maldonado, Optics Express, v. 5, 1999, 176.

The electrodes used to apply the external electric field in the EFISHtechnique can assume a variety of shapes, forms and compositions as, forexample, are found in the prior art. For instance, the electrodes can beangled or pointed to increase the electric field strength that resultsin these cases. The electrodes can be oriented in a variety of ways tothe sample; for example, the electrodes can be placed (e.g., patternedlithographically, printed, etched, etc.) on a substrate which itself isin contact with the liquid sample containing the targets and probes; or,for example, the electrodes can lie at the bottom and top of a thincavity in which the sample containing targets and probes is flowing oris held.

Non-Random Orientations and Nonlinear Activity

A non-random orientation is a necessary condition for generation of thesurface-selective nonlinear optical signal. Only the non-centrosymmetricregion of a system, is capable of generating non-linear light. Amolecule or material phase is centrosymmetric if there exists a point inspace, called the ‘center’ or ‘inversion center,’ through which aninversion (x,y,z)→(-x,-y,-z) of all atoms can be performed that leavesthe molecule or material unchanged. For example, if the molecule is ofuniform composition and spherical in shape, it is centrosymmetric.Centrosymmetric molecules or materials have no nonlinear susceptibilityor hyperpolarizability, necessary for second or higher harmonic, sumfrequency and difference frequency generation. A non-centrosymmetricmolecule or material lacks this center of inversion, and therefore canbe nonlinear-active. Non-centrosymmetric regions can be at surfaces,e.g. arrays, substrates, etc., or in bulk phase, e.g. solutions,however, a bulk phase may require the application of an electric fieldto break the symmetry of the region and render the bulk phasenonlinear-active (as in the EFISH technique).

The present invention exploits the property that any non-randomorientation of probes with respect to a surface to which they areattached or localized leads to non-random orientation of targets whenthese targets bind to the probes. As a result of the non-randomorientation of the system, a nonlinear optical technique can be used tomonitor, e.g., the binding activity at the surface. Any change in thenon-random orientation of the system, such as due to a conformationalchange on occurrence of a binding event, would modify the nonlinearoptical signal. Verification that a change in the detected nonlinearoptical signal is due to a conformational change can be accomplishedusing controls known to one skilled in the art including, for example,measuring binding of a blocking compound to the receptor—where theblocking compound is known not to produce a conformational change in thereceptor.

In an embodiment in which the probe or target, or neither, is notnatively nonlinear-active, then different types of nonlinear-activespecies can be introduced into the system to render the systemnonlinear-active. Such species that affect the one or morenonlinear-active beams emanating from a sample comprising probes andtargets include labels, decorators, indicators, modulators, inhibitors,and enhancers, as described below. When the present invention is usedwith labels or decorators, the non-random orientation of the targetproduces a non-random label or decorator orientation and this leadsdirectly to an increase in surface-selective nonlinear optical signal(e.g., intensity). Non-specific interactions of the targets withattached or localized probes (e.g., non-specific binding to probes) orof targets to regions on the surface where no probe is present (e.g.,non-specific binding to substrate or solid support) will lead to zero orto a much lower surface-selective nonlinear optical signal due todestructive interference as would be apparent to one skilled in the art.When the present invention is used with indicators, a non-random probeorientation also leads to an increase in the surface-selective nonlinearoptical signal since the surface charge density close to the surfaceplane will be larger than if the probes are randomly oriented whichresults in a lower surface electric charge density.

Nonlinear-Active Labels

A label for use in the present invention refers to a nonlinear-activemoiety, particle or molecule which can be bound, either covalently ornon-covalently, to a molecule, particle or phase (e.g., lipid bilayer)in order to render the resulting system more nonlinear optical active.Labels can be employed in the case where the molecule, particle or phase(e.g., lipid bilayer) is not nonlinear-active to render the systemnonlinear-active, or with a system that is already nonlinear-active toadd an extra manipulation parameter into the system. The exogenouslabels can be pre-attached to the molecules or particles, and anyunbound or unreacted labels separated from the labeled entities before ameasurement is made. In a specific embodiment, the nonlinear-activemoiety is attached to the target or probe molecule in vitro.Alternatively, the labels can be left in solution with probes andtargets and allowed to adsorb to some particle (e.g., an enhancer) orsurface to yield a different nonlinear-active response (i.e.,hyperpolarizability or second order susceptibility) from the boundlabels. By way of example, EFISH or Hyper-Rayleigh scattering can beused to determine if a candidate molecule or particle is nonlinearlyactive according to techniques well known in the art, where appropriatecontrols and background measurements are made in the absence of accessto the candidate nonlinear-active species. The labeling of probes withnonlinear-active labels and/or modulators of the labels allows a direct,optical means of detecting probe-target binding reactions in the caseswhere the binding reaction results in a change in orientation orconformation of the probe using a surface-selective nonlinear opticaltechnique.

In alternate embodiments of the invention, at least two distinguishablenonlinear-active labels are used. The orientation of the attached two ormore distinguishable labels would then be chosen to facilitate welldefined directions of the emanating coherent nonlinear light beam. Thetwo or more distinguishable labels can be used in assays where multiplefundamental light beams at one or more frequencies, incident with one ormore polarization directions relative to the sample, are used, with theresulting emanation of at least two nonlinear light beams.

One means of determining whether a particular molecule or particle canbe used as a nonlinear-active label is by studying it using secondharmonic generation at an air-water interface. For instance, in the caseof particles, if the particles assemble at the air-water interface in amanner which gives a net orientation of the particles (on a length scaleof the coherence length) the layer of particles will generate secondharmonic light. Another means of doing this is by measuring a sample ofa suspension of the particles and detecting the hyper-Rayleighscattering. Yet another means involves the use of EFISH to determine ifa candidate molecule or particle is nonlinearly active. The effect canbe used to measure the hyperpolarizability of molecules in solution byusing a dc field to induce alignment in the medium, and allowing SHG tobe observed. This type of measurement does not require that the particlethemselves be ordered at an interface, but does require that theparticles be nonlinear-active and thus non-centrosymmetric.

In a specific embodiment, metal nanoparticles and assemblies thereof aremodified to create biological nonlinear-active labels. The followingreferences describe the modification metal nanoparticles and assemblies:

-   J. P. Novak and D. L. Feldheim, “Assembly of Phenylacetylene-Bridged    Silver and Gold Nanoparticle Arrays”, J. Am. Chem. Soc., 2000, 122,    3979-3980.-   J. P. Novak et al., “Nonlinear Optical Properties of Molecularly    Bridged Gold Nanoparticle Arrays”, J. Am. Chem. Soc. 2000, 122,    12029-12030.-   Vance, F W, Lemon B. I., Hupp, J. T. Enormous Hyper-Rayleigh    Scattering from Nanocrystalline Gold Particle Suspensions”, J. Phys.    Chem. B 102:10091-93 (1999).

The following reference and references therein describe the techniquesavailable for creating a biological label from a synthetic dye and manyother molecules:

-   Greg T. Hermanson, Bioconjugate Techniques, Academic Press, 1996.

In a specific embodiment, nonlinear-active labels can be constructedaccording to well known procedures in the art to be photoactivated orphotomodulated with a beam of light such that, upon irradiation of thesample with a selected beam of light, the labels become nonlinearoptical active (or more or less nonlinear optical active). The beam oflight can, for example, cleave a chemical bond (e.g., using UV light),well known in the art as ‘caged’ compounds.

Modulators

Modulators include any substance (e.g., moiety, molecule, biologicalcomponent or compound) that alters the nonlinear response of anonlinear-active species when the modulator is in proximity to thenonlinear-active species, or alters the kinetic or equilibriumproperties of probe-target interactions (e.g., binding reaction).Modulators may change the rate of probe-target binding, the equilibriumconstant of probe-target binding or, in general, enhance or reduceprobe-target interactions. Examples of modulators are the following:inhibitors, drugs, small molecules, agonists and antagonists and Auparticles. Candidate modulators can be tested for their ability toperform as modulators, e.g., in a screening process.

In a specific embodiment, target-probe interactions can be measured inthe presence of some modulator of the interactions—the modulator being,for example, a small molecule, drug, or other moiety, molecule orparticle which changes in some way the target-probe interactions (e.g.,has some affinity for the probe and blocks or inhibits target binding).The modulator can be added before, during or after the time in which theprobe-target interactions occur.

Inhibitors

Inhibitors decrease or prevent probe-target interactions Inhibitors canbe any substance, e.g., moiety, molecule, compound or particle.Preferably the inhibitor competes with a known binding interactionbetween target and probe. Inhibitors are a form of modulator Inhibitorsare also referred to herein as blocking agents or blockers.

In a specific embodiment, compounds that are potential inhibitors of anagonist to a receptor are screened by testing for removal of aconformational change induced by the agonist when the receptor andagonist are also in the presence of an inhibitor candidate (the agonistcan be a natural molecule, synthetic, etc.).

Decorators

A decorator refers to a nonlinear-active substance (e.g., molecule orparticle) which can be bound to targets, probes or target-probecomplexes, and allow detection and discrimination among them. Ideally, adecorator should not appreciably alter or participate in thetarget-probe reaction itself. A decorator is distinguished from anonlinear-active label, such as a SHG-active label, in that it possessesa specific binding affinity for the target, probe, or the target-probecomplex, while an SHG-label can be attached to, for example a biologicalcomponent, via specific chemical bonds or non-specific (e.g.,electrostatic) means. A decorator can be used to detect probe-targetcomplexes by its specific binding affinity (in some art, ‘molecularrecognition) to the targets, probes or the target-probe complexes and tothereby discriminate among targets, probes and target-probe complexes ina surface-selective nonlinear optical technique. For example, adecorator which has a stronger affinity (larger binding constant) todouble-stranded DNA than single-stranded DNA can be used to detecthybridization with surface-attached probes since the amount of orienteddecorator will increase as hybridization proceeds betweensingle-stranded targets and probes. In a specific embodiment, decoratorsare constructs of a nonlinear-active species and another species, wherethe other species is a biological component (protein, antibody, etc.)and where the construct has a differential binding affinity amongprobes, targets and probe-target complexes to allow discrimination amongthem using a surface-selective nonlinear optical technique. Decoratorscan also be a non-natural molecules (e.g., synthetic chemical molecule,drug, etc.) with a nonlinear activity and which binds specifically(recognizes) targets, probes or target-probe complexes, and has adifferential binding affinity among the three to allow fordiscrimination among them using a surface-selective nonlinear opticaltechnique. In a specific embodiment, the decorator is dissolved orsuspended in a solution or aqueous phase containing the targetcomponent.

Exemplary decorator molecules or particles include, but are not limitedto, a biological component, a nucleic acid, protein, small molecule,biological cell, virus, liposome, receptor, agonist, antagonist,inhibitor, hormone, antibody, antigen, peptide, receptor, drug, enzyme,ligand, nucleoside, polynucleoside, carbohydrate, cDNA, hormone,allergen, cDNA, hapten, oligonucleotide, biotin, streptavidin,polynucleotide, oligosaccharide, peptide nucleic acid (PNA), or nucleicacid analog. In other non-limiting embodiments, decorators comprise amoiety in the family of, or that is, psoralen, ethidium bromide, methanephosphonate, phosphoramidate, propidium iodide, acridine,9-aminoacridine, acridine orange, chloroquine, pyrine, echinomycin,4′,6-diamidino-2-phenylindole, dihydro chloride (DAPI), succinimidylacridine-9-carboxylate, chloroquine, pyrine, echinomycin,4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), single-strandbinding protein (SSB), tripyrrole peptides, flavopiridol, or pyronin Y.

Indicators

The following section describes indicators in detail. Indicators includenonlinear-active molecules or particles whose nonlinear opticalproperties or orientation near a surface or interface is modulated asthe electric charge polarization, charge density or potential of thesurface is modulated. In one aspect of the invention, the charge orpotential of an interface is modulated by the binding of a target toprobes immobilized on the surface. In-another aspect, the surfaceelectric potential of a cell is changed by a change in the ion channelproperties—an opening, closing, increase or decrease in ionicpermeability in response to target (ligand) binding, for instance. Inanother aspect of the invention, an indicator serves as a marker forimaging purposes, e.g., to image cells or tissues. An indicatorpreferably does not appreciably alter or participate in the target-probereaction itself. The indicator can be dissolved or suspended in theliquid, medium, solution or aqueous phase containing the targetcomponent. An indicator preferably does not translocate into the lipidbilayer of vesicles or cells. An indicator preferably possesses freedomof movement to respond to changes in surface electric charge density orpotential.

Measuring the nonlinear optical response of a glass-solvent orglass-water interface, in the presence of dissolved or suspendedindicators in the water or solvent, is one means of assaying whether acandidate molecule would function as an indicator. Since glass carries anet negative charge, a candidate molecule can function as an indicatorif the intensity of the nonlinear optical radiation generated at theinterface in the presence of the molecule is greater than the backgroundsignal in its absence. Another means of assaying for a candidatemolecule's ability as an indicator is by measuring the intensity ofnonlinear optical radiation generated by a semiconductor-liquidinterface as a function of applied voltage (and hence surface electriccharge density) between the semiconductor and the bulk of the liquid.Yet another means is to measure the hyper-rayleigh scattering (HRS) froma solution or suspension of the indicator candidates, since if HRS isgenerated and the candidate itself is charged or dipolar, it shouldserve as an indicator.

Oxazole dye 4-[5-methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate(also known as 4PyMPO-MeMs) is an indicator that can be used, that isstrongly second harmonic-active and chemically stable at neutral pH(Salafsky and Eisenthal, Chemical Physics Letters 2000, 319, 435-439).Furthermore, the Stokes shift of the fluorescence which results fromtwo-photon absorption is large so that the second harmonic beam canreadily be separated from the fluorescence. Other dyes in this familyhave similar properties (J. H. Hall et al., “Syntheses and PhotophysicalProperties of Some 5(2)-Aryl-2(5)-(4-pyridyl)oxazoles and RelatedOxadiazoles and Furans”, J. Heterocyclic Chem. 29, 1245 (1992)). Theseand other molecules, or assemblies of the molecules, can be used asindicators in the present invention. Such molecules include, but are notlimited to:

-   5-(4-methoxyphenyl)-2-(4-methoxyphenyl)-2-(4-pyridyl)oxazole-   2-(4-methoxyphenyl)-5-(4-pyridyl)oxazole-   2-(4-methoxyphenyl)-5-(4-pyridyl)oxadiazole-   2-(4-methoxyphenyl)-5-(4-pyridyl)furan-   2-(4-pyridyl)-4,5-dihydronapthol[1,2-d]-1,3-oxazole-   5-Aryl-2-(4-pyridyl)-4-R-oxazole where R is a hydrogen atom, methyl    group, ethyl group or other alkyl group.-   2-(4-pyridyl)cycloalkano[d]oxazole-   2-(4-pyridyl)phenanthreno[9,10-d]-1,3-oxazole-   6-Methoxy-4,4-dimethyl-2-(4-pyridyl)indeno[2,1-d]oxazole-   4,5-Dihydro-7-methoxy-2-(4-pyridyl)napthol[1,2-d]-1,3-oxazole

Other molecules or molecules of the following families which can be usedas indicators, include, but are not limited to:

Merocyanines

Stilbenes

Indodicarbocyanines

Hemicyanines

Stilbazims

Azo dyes

Cyanines

Stryryl-based dyes

Methylene blue

Diaminobenzene compounds

Polyenes

Diazostilbenes

Tricyanovinyl aniline

Tricyanovinyl azo

Melamines

Phenothiazine-stilbazole

Polyimide

Sulphonyl-substituted azobenzenes

Indandione-1,3-pyidinium betaine

Fluorescein

Benzooxazole

Perylene

Polymethacrylates

Oxonol

Derivatized Particle Indicators

A solid microparticle or a nanoparticle of size nanometers to microns inscale including, but not limited to, a sphere (latex, polystyrene,silica, etc.) or a strip, offers a surface area which can be derivatizedwith a nonlinear-active moiety via chemical or electrostatic means sothat the entire object has a much higher hyperpolarizability than can beobtained otherwise. For instance, nonlinear-active dyes can be orderedon silica bead surfaces via electrostatic interactions (dye ispositively charged, silica surface is negatively charged) and the entirebead, if derivatized with target-reactive linkers, can then function asan indicator. If the nonlinear-active moieties can be aligned on thesolid surface so that phase interference between moieties is small, theoverall hyperpolarizability will scale nonlinearly (e.g., quadratically)in their number. The solid particle can vary in shape and its size canrange from nanometers to microns in scale. Examples of the particles tobe used include, but are not limited to, polystyrene beads and silicabeads, both readily commercially available.

a. Covalent Attachment

The solid particles to be used as indicators can be surface-derivatizedusing a variety of chemistries available in the prior art.Nonlinear-active moieties can be covalently coupled either to the solidparticles or to a derivatized layer.

For instance, polystyrene beads can be derivatized with dextran, lactoseor amines (the latter case for example, via chloromethyl groups withethylenediamine). Silica can be derivatized using organofunctionalsilanes, for example using trichlorosilanes or other functional silanes(such as methoxy, amine, or other functional groups), to producesurfaces with a variety of chemical functionalities. The surfaces of thederivatized beads can then be reacted with a nonlinear-active moiety viaappropriate chemistry to produce the indicator.

b. Electrostatic Attachment

Nonlinear-active moieties can also be electrostatically bound to amicron- or nanometer-sized particle surface to produce indicators withlarge hyperpolarizabilities. A charged nonlinear-active moiety, anorganic dye for example, can be oriented at a counter-chargedmicroparticle surface, thus allowing for a net hyperpolarizability ofthe object when using an appropriate geometry. An example of anappropriate geometry is a microparticle sphere where the diameter isapproximately the wavelength of the fundamental light, i.e. from tens ofnanometers to microns so that destructive phase interference betweennonlinear-active moieties on opposing faces of the sphere is minimized.The hyperpolarizability of each dye at the spheres's surface, whenintegrated across the entire surface of the sphere of ˜wavelength oflight size, is large and positive. By way of example, but notlimitation, the following procedure can be used. Silica beads (˜200 nm,roughly spherical) are reacted with a low concentration of3-aminopropyltrimethoxysilane or 3-aminooctyltrimethoxysilane so thatonly-5-10% of the surface silanols become covalently coupled to thesilane agent. These amine groups are then reacted with theamine-reactive homobifunctional crosslinker disuccinimidyl glutarate(DSG, Pierce Chemical) to create amine-reactive linkers on-5-10% of thebead surface. The beads are then incubated with4-[5-methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate (also knownas 4PyMPO-MeMs), a positively charged dye which binds electrostaticallyto the charged silanols on the surface and orients to some degree. Theexcess dye is removed from the beads by centrifugation. Theelectrostatic adsorption can be sufficiently high in some cases toimmobilize the charged dye, even in the absence of a bulk concentrationof it. Many nonlinear-active species are known in the art that can beused and include, but are not limited to, the following and theirderivatives:

Oxazole or oxadizole molecules

5-aryl-2-(4-pyridyl)oxazole

2-aryl-5-(4-pyridyl)oxazole

2-(4-pyridyl)cycloalkano[d]oxazoles

Merocyanines

Stilbenes

Indodicarbocyanines

Hemicyanines

Stilbazims

Azo dyes

Cyanines

Stryryl-based dyes

Methylene blue

Diaminobenzene compounds

Polyenes

Diazostilbenes

Tricyanovinyl aniline

Tricyanovinyl azo

Melamines

Phenothiazine-stilbazole

Polyimides

Sulphonyl-substituted azobenzenes

Indandione-1,3-pyidinium betaine

Fluoresceins

Benzooxazoles

Perylenes

Polymethacrylates

Oxonols

Thiophenes

Bithiophenes

In evaluating whether a species may be nonlinear-active, the followingcharacteristics can indicate the potential for nonlinear activity: alarge difference dipole moment (difference in dipole moment between theground and excited states of the molecule), a large Stokes shift influorescence, an aromatic or conjugated bonding character. In furtherevaluating such a species, an experimenter can use a simple techniqueknown to those skilled in the art to confirm the nonlinear activityusing, for example, detection of SHG from an air-water interface or fromEFISH in the absence and presence of the species in question in amedium. Once a suitable nonlinear-active species has been selected forthe experiment at hand, the species can be conjugated, if desired, to aspecies with specificity to a biological target to produce a targetingconstruct used in the surface-selective nonlinear optical detection orimaging technique.

Enhancers

An enhancer as used herein refers to a substance (e.g., moiety, moleculeor particle) which can enhance (increase) the cross-section of anonlinear-active substance (e.g., moiety, molecule or particle) whenplaced near to it (e.g., increase the intensity of second harmonicradiation generated). Examples of the enhancement effect referred to inthe art include ‘resonance enhancement’ and ‘surface enhancement.’Enhancement of the nonlinear-active cross-section moiety, molecule orparticle can occur via a resonance with an electronic transition orplasmon resonance of the enhancer. The addition of an enhancer onto, ornear to, a molecule or surface, can result in the enhancer coupling tothe molecule or surface, e.g., covalently, electrostatically,non-covalently, etc., or the enhancer is not coupled directly to themolecule or surface, but rather is near to the molecule or surface,e.g., enhancer adsorption on to a cell surface, causing the enhancer toincrease the nonlinear response of the label on the probes.

The following (and references therein) describe the production, designand use of resonance-enhancing particles, such as metal nanoparticles,for nonlinear optical processes (SHG, Surface-enhanced resonance Raman,etc.): S, Nie and S. Emory, Science, 1997, 75, 1102; P. V. Kamat, M.Flumiani, G. V. Hartland, J. Phys. Chem. B, 1998, 102, 3123; H.Ditlbacher et al., Appl. Phys. B, 2001, DOI: 10, 1007/s003400100700; C.Sonnichsen et al., Appl. Phys. Lett., 2000, 77, 2949; B. Lamprecht etal., Appl. Phys. B, 1997, 64, 269; J. P. Novak, J. Am. Chem. Soc., 2000,122, 12029; S. R. Emory, W. E. Haskins, S, Nie, J. Am. Chem. Soc. 1998,120, 8009; W. P. McConnell et al., J. Phys. Chem. B 2000, 104, 8925; F.W. Vance, B. I. Lemon, J. T. Hupp, J. Phys. Chem. B 1998, 102, 10091; P.Galletto et al., J. Phys. Chem. B 1999, 103, 8706; S. R. Emory, S, Nie,J. Phys. Chem. B 1998, 102, 493

The presence of the resonance-enhancing or surface-enhancing speciesserves to increase the nonlinear-active cross-section of samples.Examples of resonance-enhancing species in the art are the following:metal or metallic (e.g., gold and silver) nanoparticles or colloidalparticles, metal-coated particles (e.g., silver-coated latexnanospheres), aggregates or clusters of any of the aforementioned,rationally-designed clusters, chains or aggregates of the aforementioned(e.g., for symmetry-breaking: non-centrosymmetric aggregates, particlesor clusters), etc.

In some instances, experimentation may be required to determine theoptimal labeling strategy and/or use of enhancers or decorators for agiven measurement. For instance, the coupling chemistry, reactionconditions, etc. may be adjusted empirically to determine the optimallabeling strategy. Candidates for an enhancer can be tested for theireffect on a nonlinear-active species by measuring, for example, the SHGintensity of a nonlinear-active species in the absence and presence ofthe candidate enhancer (the enhancer can be attached to thenonlinear-active label, through a linker if necessary; or the enhancercan be brought into proximity to the label, e.g. by virtue of theprobe-target reaction). The enhancer's effect on a nonlinear-activelabel can be made at an interface (e.g., air-water or solid-liquid) orin bulk phase under the application of an electric field (EFISH).

Molecular Beacon Analogues

The nonlinear activity of a system can also be manipulated through theintroduction of nonlinear analogues to molecular beacons, that is,molecular beacon probes that have been modified to incorporate anonlinear-active label (or modulator thereof) instead of fluorophoresand quenchers. These nonlinear optical analogues of molecular beaconsare referred to herein as molecular beacon analogues (MB analogues orMBA). The MB analogues to be used in the invention can be synthesizedaccording to procedures known to one of ordinary skill in the art.

In specific embodiments, the MB analogue probes can be used according tothe present invention as hybridization probes that can report thepresence of complementary nucleic acid targets without having toseparate probe-target hybrids from excess probes in hybridization assaysand without the need to label the targets. Target labeling is not onlytime-consuming, but it can change the levels of targets originallypresent in a sample. MB analogue probes can also be used for thedetection of RNAs within living cells, for monitoring the synthesis ofspecific nucleic acids in sealed reaction vessels, and for theconstruction of self-reporting oligonucleotide arrays. They can be usedto perform homogeneous one-tube assays for the identification ofsingle-nucleotide variations in DNA and for the detection of pathogensor cells immobilized to surfaces for interfacial detection.

FIGS. 20A and 20B illustrate an embodiment of a MB analogue probe. Aspecies with a hyperpolarizability capable of generating nonlinearoptical radiation in response to illumination with one or morefundamental beams is attached to one end of a probe or at one location(e.g., a dye molecule, see FIG. 20B). At the other end of the probe orin another location in proximity to the nonlinear-active label, aspecies capable of increasing or decreasing the nonlinear opticalradiation generated by a nonlinear-active label when the two species arein proximity is attached (e.g., a 10 nm gold particle, see FIG. 20B).The attachment can be via a covalent bond or through some other wellknown means in the art. In a specific embodiment, the nonlinear-activelabel is an organic dye with a hyperpolarizability such as the wellknown oxazole dyes and their derivatives. The oxazole dyes and theirderivatives are commercially available from Molecular Probes (Eugene,Oreg.) for attachment to a variety of probes including nucleic acidsusing amine and sulfhydryl chemistries. A species such as a goldnanoparticle, well known for its ability to enhance the nonlinearoptical radiation generated by a nonlinear-active species to which it isin proximity, can be used as the species capable of modulating thenonlinear optical radiation generated by the label. In specificembodiments, the MB analogue probes can be immobilized to a solidsurface such as a planar glass surface or to the surface ofmicrospheres, or the MB analogue probes can be used in homogeneoussolution and detected using an applied electric field in an EFISHmeasurement. If the MBAs are immobilized to a surface, thenonlinear-active species becomes at least partially oriented at thesurface and satisfies the non-centrosymmetric condition required forsurface-selective nonlinear optical techniques.

The Au nanoparticle can enhance the intensity of nonlinear opticalradiation, such as second harmonic generation scattered by the oxazoledye by several orders of magnitude when the nanoparticle and oxazole dyeare in proximity to each other. Upon hybridization of the probe to acomplementary target, the intensity of the nonlinear optical radiationdecreases and this decrease can be quantitatively related to the amountof probe-target hybridization. The sensitivity of the technique isdetermined by, among other factors, the background nonlinear opticalsignal before hybridization occurs. The sequences of various targetsused for testing the degree of hybridization to the probe in FIG. 20Bare Target 1-4 in FIG. 20 (SEQ ID NOs: 1-4 respectively).

The present invention can be used for detection of single nucleotidepolymorphisms (SNPs) in target samples because the MBA probes are highlyselective in their binding of targets and a one base pair difference insequence between probes and targets will yield a much reducedhybridization affinity compared with a target that is perfectlycomplementary. The MBA probe can act also as a label.

Assaying Probe-Target Interactions

Throughout the description of the present invention test molecules arealso referred to as “targets,” and candidate binding partners are alsoreferred to herein as “probes.” The following are examples of the typesof probe-target interactions that can be assayed according to thepresent invention:

-   -   i) Probe-target binding that results in a conformational change        in the probe, target or both.    -   ii) Probe-target binding that results in a change in the dipole        moment of a nonlinear-active species, said species being the        probe, target or both, as a result of probe-target binding.

Probe-target binding can also result in a combination of bothconformational and dipole moment changes.

Furthermore, detection of the probe-target interactions can occur at aninterface, in bulk phase (homogeneous phase), or in regions thatcomprise a combination of interface and bulk.

The present invention can be applied to an ensemble of molecules or to asingle molecule—i.e., ensemble reaction measurements or asingle-molecule reaction measurement.

In a preferred embodiment, the target is a G protein-coupled receptor(GPCR). GPCRs are one class of proteins that undergo a conformationalchange when activated by a ligand and are thus amenable to study usingthe present invention. In this case, if the GPCR is not intrinsicallynonlinear-active, the protein is labeled using a nonlinear-active label,and the—conformational change is detected or queried for via a change inthe orientation of the nonlinear-active label. The GPCRs can be attachedto a surface and the conformational change that results when a ligandactivates the receptor causes a change in the orientation of the labeland thus a change in properties of the nonlinear optical beams (e.g.,second harmonic generation) such as intensity, wavelength orpolarization. A background signal can be measured before exposure of thesample to a ligand, if desired.

In some cases, binding of a component to a receptor will lead to achange in measured nonlinear optical properties even though the receptoris not activated. For example, this can be due to an interaction betweenthe component and the receptor in the bound complex which alters theorientation of a label attached to the receptor. A control can beperformed, if desired, to assign measured changes in nonlinear opticalproperties to binding or activation of components to a given receptor.For example, a component which is known to bind to a given receptor butnot to produce a conformational change can be used as a control of thelabel reaction; if any measured change in label orientation is due onlyto receptor activation, the position of the label can be changed bychanging the conjugation chemistry of the label and/or geneticallymodifying the receptor to introduce new labeling sites.

For example, receptors can be labeled with a nonlinear-active label thatpossesses, on average, an orientation (or orientation ofhyperpolarizability) of some angle θ with respect of the surface plane(e.g., host cell membrane). Upon binding and/or activation to someligand, the average label angle can shift to angle P with respect to thesurface plane. Even small shifts in angle as a result of receptorbinding or activation can cause substantial changes in a property of themeasured nonlinear optical light (e.g., intensity of the nonlinearlight). For example, if the intensity of the nonlinear optical lightgenerated is proportional to the component of the hyperpolarizabilitythat is normal to the membrane plane, then the percentage change ofintensity upon a shift is: [cos(θ)/cos(β)]² with the nonlinear intensitydependent quadratically on the normal component of thehyperpolarizability. For example, assuming a delta function inhyperpolarizability orientation, if θ→β is a change of 25 degrees to 30degrees, the nonlinear optical intensity will decrease about 9%.

In some instances, some experimentation may be required to determine theoptimal labeling strategy and/or use of enhancers or decorators for agiven measurement. For instance, the coupling chemistry, reactionconditions, etc. may need to be adjusted empirically to determine theoptimal labeling strategy.

Alternatively, the target molecules can be solubilized and, using theirintrinsic dipole moments, poled by an electric field in solution phase;if the target possesses a hyperpolarizability (i.e., isnonlinear-active, either intrinsically or made so by attachment of anonlinear-active label), a nonlinear optical signal will be generatedvia EFISH technique, and this signals serves as the background. Uponaddition of a ligand (i.e., a probe) that activates the target molecule,a conformational change will occur. If this conformational change doesnot result in an appreciable dipole moment change in the targetmolecule, the number and net orientation of the target molecules willnot change appreciably. If, however, this conformational change ispassed to the nonlinear-active moiety, its overall orientation willchange, resulting in a change in measured nonlinear optical properties.If the binding of a ligand to the target molecules results in anappreciable dipole moment change of the nonlinear-active targetmolecule, the number of aligned target molecules in the applied fieldwill change, resulting in-change in measured nonlinear opticalproperties. The number of aligned molecules in an applied electric fieldin solution is dependent on the dipole moment of the molecules. Anequation used to model the dependence is the Langevin equation:N═N_(o)exp(−[−μE/kT) where N_(o) is the number of aligned species, μ istheir dipole moment, E is the electric field magnitude parallel to thedipole moment, N_(o) the number of molecules exposed to the field, k isthe Boltzmann constant and T is the temperature in Kelvin. Changes indipole moment as a result of probe-target interactions can lead to largechanges in the number of aligned molecules and thus large changes in theintensity of nonlinear optical light generated by the molecules. Forexample, a probe with a nonlinear-active label and a given dipole momentbinds to a target; the resulting probe-target complex with a larger orsmaller dipole moment leads to a change in intensity of the generatednonlinear light. Measured changes as a result of binding can also beused to calculate binding conformations between molecules if theposition and amount of charge is well known on each molecule (e.g., asis often the case with proteins, whose crystal structure is known).

The above illustrations are exemplary of any probe-target interactionthat results in a change in dipole moment or conformational change. Ionchannel proteins are examples of another important class of proteinsthat undergo conformational change in response to activation and arealso amenable to study using the present invention.

Surface-selective nonlinear optical techniques are also coherenttechniques, meaning that the fundamental and nonlinear beams havewell-defined phase relationships, and the wavefronts of a nonlinear beamin a macroscopic sample (within the coherence length) are in phase.These properties offer a number of advantages useful for surface orhigh-throughput studies in which, for example, either a single surfaceor a microarray surface is examined. An apparatus using nonlinearoptical surface-selective-based detection, such as with second harmonicgeneration, requires minimal collection optics since generation of thenonlinear light only occurs at the interface and thus, in principle,allows extremely high depth discrimination and fast scanning

Probe-target interactions (e.g., a binding reaction, a conformationalchange, etc.) can be correlated with the present invention to thefollowing measurable information, for example:

-   -   i) the intensity of the nonlinear or fundamental light.    -   ii) the wavelength or spectrum of the nonlinear or fundamental        light.    -   iii) position of incidence of the fundamental light on the        surface or substrate (e.g., for imaging).    -   iv) the polarization of the nonlinear light.    -   v) the time-course of i), ii), iii) or iv).    -   vi) one or more combinations of i), ii), iii), iv) and v).

The advantages of the present invention are enumerated as follows:

-   -   i) Sensitive and direct dependence on the orientation and/or        dipole moment of the nonlinear-active species in a sample,        useful for detection of conformational changes in probes and        binding that results in an appreciable change in the dipole        moment of the nonlinear-active species (i.e., probe, target or        both).    -   ii) Higher signal to noise (lower background) than        fluorescence-based detection since surface-selective nonlinear        optical light is generated only at surfaces that create a        non-centrosymmetric situation, or in homogeneous phase under        application of an electric field to induce the        non-centrosymmetry surface-selective nonlinear optical light        detection of a surface has a very narrow ‘depth of field’.        Sources of fluorescence in fluorescence-based detection schemes        include that from materials in the field of view but not in the        focal plane, autofluorescence, and contamination of the emitted        fluorescence with stray excitation light; these are not sources        of background nonlinear optical radiation.    -   iii) The nonlinear optical technique is useful when the presence        of a liquid solution is required for the measurement, i.e. where        the binding process can be obviated or disturbed by a wash-away        step. This aspect of the invention can be useful for equilibrium        measurements (free energy, binding constants, etc.), which        require the presence of bulk species or kinetics measurements        with measurements made over a period of time.    -   iv) Lower photobleaching and heating effects than those that        occur in fluorescence—the two-photon absorption cross-section is        much lower than the one-photon cross-section in a molecule and        the nonlinear optical technique involves scattering, not        absorption.    -   v) A minimum of collection optics is needed and higher signal to        noise is expected since the fundamental and nonlinear beams        (e.g., second harmonic) have well-defined incoming and outgoing        directions with respect to the interface. This is advantageous        compared to fluorescence-based detection in which the        fluorescence is emitted isotropically and there may be a large        auto-fluorescence background out of the plane of interest (e.g.,        the interface containing the probes).    -   vi) Ease of use with beads, biological cells, liposomes or other        particles whose non-planar surface makes an interface with the        supporting medium, solution, etc.    -   vii) Convenience of discriminating between binding of targets to        probes from actual activation of probes (e.g., a receptor) by a        target.    -   viii) The binding process between probes and targets can be        performed in the presence of one or more small molecules, drugs,        blocking agents, or other components which affect properties of        the probe-target binding process, e.g. equilibrium constants,        kinetics of binding, etc.

In an embodiment, using surface arrays, arrays can be constructedaccording a plurality of methods found in the art. For DNA microarrays,most are prepared with one of three non-standard approaches (S. C.Case-Green et al., Curr. Opin. Chem. Biol. 2 (1998), 404): Affymetrix,Inc. probe arrays are prepared using patterned, light-directedcombinatorial chemical synthesis (S. A. Fodor, Science 277 (1997), 393);spotted arrays can be made according to D. H. Duggan et al., NatureGenet. 21 (Suppl.) (1999), 10; M. Schena et al., Science 270 (1995),467; P. O. Brown and D. Botstein, Nature Genet. 21 (Suppl.) (1999), 33;and L. McAllister et al., Am. J. Hum. Genet. 61 (Suppl.) (1997), 1387;ink jet techniques can also be used to synthesize oligonucleotides baseby base through sequential solution-based reactions on an appropriatesubstrate (A. P. Blanchard et al., Biosens. And Bioelectron. 11 (1996)687-relevant portions of all of which references are incorporated byreference herein).

For example, nucleic acid, oligo- or nucleotide arrays can beconstructed according to U.S. Pat. No. 6,110,426, U.S. Pat. Nos.5,143,854 6,110,426-relevant portions of which are incorporated byreference herein, U.S. Pat. No. 5,143,854-relevant portions of which areincorporated by reference herein or Fodor et al., “Light-directedSpatially-addressable Parallel Chemical Synthesis,” Science, 1991, 251,767-773. Soluble protein arrays can be constructed according to R.Ekins, F. W. Chu, Trends in Biotechnology, 1999, 17, 217, relevantportions of which are incorporated by reference herein. Membraneproteins arrays can be constructed by micropatterning of fluid lipidmembranes according, for example, to the method of Groves, J. T., Ulman,N., Boxer, S. G., “Micropatterning fluid bilayers on solid supports”,Science, 1997, 275, 651-3 (relevant portions of which are incorporatedby reference herein). The array substrate can be composed of glass,silicon, indium tin oxide, or any other substrate known in the art. Thesurface array under study can contain physical barriers between elementsso that the elements (and their biomolecules) can remain in isolationfrom each other during a chemical reaction step. The array locations canconsist of different probes, the same probes everywhere, or somecombination thereof. The array can also be constructed on the undersideof a prism allowing for total internal reflection of the beam andevanescent generation of the nonlinear light. Or an array substrate canbe brought into contact with a prism with the same result.

An electrophoretic system can also be used in conjunction with thesurface array, for example to deliver reagents or biological componentsto one or a plurality of locations using flow channels ormicrocapillaries. The sample can include an array of microcapillarychannels, each distinct from the other and each allowing a target-probereaction to occur; the imaging technique would then consist of arrayelements, each one a microcapillary channel or reaction chamber intowhich the channel feeds or drains.

The polarization of the fundamental and nonlinear beams can be selectedwith polarizing optics elements. By analyzing the intensity of thenonlinear beam as a function of fundamental and nonlinear polarization,more information (e.g., higher signal to noise) about the probe-targetcomplexes can be obtained. Furthermore, by selecting and analyzing thepolarization of the fundamental or nonlinear optical radiation,background radiation can be reduced or signal intensity enhanced.

Detection can be accomplished with the use of multiple internalreflection plates (N. J. Harrick, “Internal Reflection Spectroscopy”,John Wiley & Sons, Inc., New York, 1979—relevant portions of which areincorporated by reference herein) allowing the fundamental beam to makemultiple contacts with the array surface, thus increasing the intensityof the generated nonlinear light. Another alternative is to construct anoptical cavity with the array surface on one side and a lossy coupler atone end to permit the output coupling of the nonlinear light, creatingan optical microcavity which would allow the buildup of very highintensities under resonance and thus increase the amount of nonlinearlight generated.

Polynucleotide arrays can be used as probes. Where oligonucleotides aretargets or probes, preferably a nonlinear-active label is attached tothe 5′ or 3′ termini. There are many linking moieties and methodologiesfor attaching molecules which can be nonlinear-active labels to the 5′0or 3′ termini of oligonucleotides, as exemplified by the followingreferences: Eckstein, editor, Oligonucleotides and Analogues: APractical Approach (IRL Press, Oxford, 1991); Zuckerman et al., NucleicAcids Research, 15: 5305-5321 (1987) (3′ thiol group onoligonucleotide); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′ sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′ phosphoamino groupvia Aminolink™ II available from Applied Biosystems, Foster City,Calif.) Stabinsky, U.S. Pat. No. 4,739,044 (3′ aminoalkylphosphorylgroup); Agrawal et al., Tetrahedron Letters, 31: 1543-1546 (1990)(attachment via phosphoraridate linkages); Sproat et al., Nucleic AcidsResearch, 15: 4837 (1987) (5′ mercapto group); Nelson et al., NucleicAcids Research, 17: 7187-7194 (1989) (3′ amino group); and the like,relevant portions of which are incorporated by reference herein.

Preferably, commercially available linking moieties are employed thatcan be used to a label to an oligonucleotide during synthesis, e.g.,available from Clontech Laboratories (Palo Alto, Calif.). In a specificembodiment, rhodamine and fluorescein dyes can be conveniently attachedto the 5′ hydroxyl of an oligonucleotide at the conclusion of solidphase synthesis by way of dyes derivatized with a phosphoramiditemoiety, e.g., Woo et al., U.S. Pat. No. 5,231,191; and Hobbs, Jr., U.S.Pat. No. 4,997,928, relevant portions of which are incorporated byreference herein.

Preferable, the oligonucleotides are present on arrays.

Protein arrays can be used to determine whether a given target proteinbinds to the immobilized probe protein on the surface; these arrays canalso be used to study small molecule binding to the probe proteins.Protein arrays can be prepared by the method of G. MacBeath and S. L.Schreiber, “Printing Proteins as Microarrays for High-ThroughputFunction Determination”, Science 2000, 289, 1760-1763, for example, todetermine whether a given target protein binds to the immobilized probeprotein on the surface.

The surface on which the probes are formed may be composed from a widerange of material, either biological, non-biological, organic,inorganic, or a combination of any of these, existing as particles,strands, precipitates, gels, sheets, tubing, spheres, containers,capillaries, pads, slices, films, plates, slides, etc. The surface mayhave any convenient shape, such as a disc, square, sphere, circle, etc.The surface is preferably flat but may take on a variety of alternativesurface configurations. For example, the surface may contain raised ordepressed regions on which a sample is located. The surface and itssurface preferably form a rigid support on which the sample can beformed. The surface and its surface are also chosen to provideappropriate light-absorbing characteristics. For instance, the surfacemay be a polymerized Langmuir Blodgett film, functionalized glass, Si,Ge, GaAs, GaP, Si.sub.xO.sub.y, Si.sub.xN.sub.y, modified silicon, orany one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, orcombinations thereof. Other surface materials will be readily apparentto those of skill in the art upon review of this disclosure. In apreferred embodiment the surface is flat glass or silica.

According to some embodiments, the surface of the substrate is etchedusing well known techniques to provide for desired surface features. Forexample, by way of the formation of trenches, v-grooves, mesastructures, or the like, the synthesis regions may be more closelyplaced within the focus point of impinging light. The surface may alsobe provided with reflective “mirror” structures for maximization ofemission collected therefrom.

The chemical identity of the probes (e.g., protein structure oroligonucleotide sequence) can vary from site to site across the solidsurface, or the same probe can uniformly cover the surface. Targets canbe of a single identity or a combination of targets with differentidentities.

In a specific embodiment, the kinetics of probe-target binding reactionsare measured as a function of target concentration. In this embodiment,the time course of the intensity and/or spectrum of the nonlinearoptical light are measured. The measured information is converted into atime course of bound target concentration (e.g., probe-targetconcentration in mM/s or EM/s). Drugs or modulators of the probe-targetbinding equilibrium or kinetic rate of formation can be used so as tocompare the effect of the added substance on the probe-target reactions.

Various art not involving the use of a surface-selective nonlinearoptical technique contains relevant portions for the present inventionand the following exemplary list and their references therein isreferenced herein: King et al., U.S. Pat. No. 5,633,724 for the scanningand analysis of the scans; Fork et al., U.S. Pat. No. 6,121,983 for themultiplexing of a laser to produce a laser array suitable for scanning;Foster, U.S. Pat. No. 5,485,277; Fodor et al., U.S. Pat. No. 5,324,633and Fodor et al., U.S. Pat. No. 6,124,102 for a substrate containing anarray of attached probes and for the analysis of scans to determinekinetic and equilibrium properties of a binding reaction between probesand targets; Kain et al., U.S. Pat. No. 5,847,400 for laser scanning ofa substrate; King et al., U.S. Pat. No. 5,432,610 for an opticalresonance cavity for power build-up; Walt et al., U.S. Pat. No.5,320,814, Walt et al., U.S. Pat. No. 5,250,264, Walt et al., U.S. Pat.No. 5,298,741, Walt et al., U.S. Pat. No. 5,252,494, Walt et al., U.S.Pat. No. 6,023,540, Walt et al., U.S. Pat. No. 5,814,524, Walt et al.,U.S. Pat. No. 5,244,813 for fiber-optic-based apparatus; Fiekowsky etal., U.S. Pat. No. 6,095,555 for imaging and software-based analysis ofimages; Stem et al., U.S. Pat. No. 5,631,734 for data acquisition;Stimson et al., U.S. Pat. No. 6,134,002 for confocal imaging techniques;Sampas, U.S. Pat. No. 6,084,991 for CCD-based imaging techniques; Stemet al., U.S. Pat. No. 5,631,734 for photolithographical preparation ofprobes attached to surfaces; Shalon et al., U.S. Pat. No. 6,110,426 formethods and apparatus for creating attached probes on a surface;Slettnes, U.S. Pat. No. 6,040,586 for position-based scanningtechniques; Trulson et al, U.S. Pat. No. 6,025,601 for methods ofimaging probe-target binding on a surface.

Cells Attached to Surfaces and Microarrays of Cells

This section outlines some of the methods concerned with interfacingbiological cells with surfaces and fabricating arrays of biologicalcells on surfaces, which can be used in the assays of the presentinvention. Many methods have been described for making uniformmicro-pattered arrays of cells for other applications, using for examplephotochemical resist-photolithograpy. (Mrksich and Whitesides, Ann. Rev.Biophys. Biomol. Struct. 25:55-78, 1996). According to this photoresistmethod, a glass plate is uniformly coated with a photoresist and a photomask is placed over the photoresist coating to define the “array” orpattern desired. Upon exposure to light, the photoresist in the unmaskedareas is removed. The entire photolithographically defined surface isuniformly coated with a hydrophobic substance such as an organosilanethat binds both to the areas of exposed glass and the areas covered withthe photoresist. The photoresist is then stripped from the glasssurface, exposing an array of spots of exposed glass. The glass platethen is washed with an organosilane having terminal hydrophilic groupsor chemically reactable groups such as amino groups. The hydrophobicorganosilane binds to the spots of exposed glass with the resultingglass plate having an array of hydrophilic or reactable spots (locatedin the areas of the original photoresist) across a hydrophobic surface.The array of spots of hydrophilic groups provides a substrate fornon-specific and non-covalent binding of certain cells, including thoseof neuronal origin (Klienfeld et al., J. Neurosci. 8:4098-4120, 1988).Reactive ion etching has been similarly used on the surface of siliconwafers to produce surfaces patterned with two different types of texture(Craighead et al., Appl. Phys. Lett. 37:653, 1980; Craighead et al., J.Vac. Sci. Technol. 20:316, 1982; Suh et al. Proc. SPIE 382:199, 1983).

In another method based on specific yet non-covalent interactions,photoresist stamping is used to produce a gold surface coated withprotein adsorptive alkanethiol. (Singhvi et al., Science 264:696-698,1994). The bare gold surface is then coated with polyethylene-terminatedalkanethiols that resist protein adsorption. After exposure of theentire surface to laminin, a cell-binding protein found in theextracellular matrix, living hepatocytes attach uniformly to, and growupon, the laminin coated islands (Singhvi et al. 1994). An elaborationinvolving strong, but non-covalent, metal chelation has been used tocoat gold surfaces with patterns of specific proteins (Sigal et al.,Anal. Chem. 68:490-497, 1996). In this case, the gold surface ispatterned with alkanethiols terminated with nitriloacetic acid. Bareregions of gold are coated with tri(ethyleneglycol) to reduce proteinadsorption. After adding Ni²⁺, the specific adsorption of fivehistidine-tagged proteins is found to be kinetically stable.

More specific uniform cell-binding can be achieved by chemicallycrosslinking specific molecules, such as proteins, to reactable sites onthe patterned substrate (Aplin and Hughes, Analyt. Biochem. 113:144-148,1981). Another elaboration of substrate patterning optically creates anarray of reactable spots. A glass plate is washed with an organosilanethat chemisorbs to the glass to coat the glass. The organosilane coatingis irradiated by deep UV light through an optical mask that defines apattern of an array. The irradiation cleaves the Si—C bond to form areactive Si radical. Reaction with water causes the Si radicals to formpolar silanol groups. The polar silanol groups constitute spots on thearray and are further modified to couple other reactable molecules tothe spots, as disclosed in U.S. Pat. No. 5,324,591, incorporated byreference herein. For example, a silane containing a biologicallyfunctional group such as a free amino moiety can be reacted with thesilanol groups. The free amino groups can then be used as sites ofcovalent attachment for biomolecules such as proteins, nucleic acids,carbohydrates, and lipids. Other methods for patterning the adhesion ofmammalian cells to surfaces using self-assembled monolayers on a surfaceinclude Lopez et al., “Convenient Methods for Patterning the Adhesion ofMammalian Cells to Surfaces Using Self-Assembled Monolayers ofAlkanethiolates on Gold,” J. Am. Chem. Soc., 1993, 115, 5877-5878,Georger et al., “Coplanar Patterns of Self-assembled Monolayers forSelective Cell-adhesion and Outgrowth,” Thin Solid Films 210 (1-2):716-719 Apr. 30, 1992, and Spargo et al., PNAS 91 (23): 11070-11074(1994).

The non-patterned covalent attachment of a lectin, known to interactwith the surface of cells, to a glass substrate through reactive aminogroups has been demonstrated (Aplin and Hughes, Analyt. Biochem.113:144-148, 1981). The optical method of forming a uniform array ofcells on a support requires fewer steps and is faster than thephotoresist method, (i.e., only two steps), but it requires the use ofhigh intensity ultraviolet light from an expensive light source.

Cells can also be cultured or grown on surfaces that require noadditional derivatization. Surfaces of this type well known in the artinclude Becton-Dickinson Falcon plates and others.

In all of these methods the resulting array of cells or surface-attachedcell layer is uniform. In the photoresist method, cells bind to thearray of hydrophilic spots and/or specific molecules attached to thespots, which, in turn, bind cells. Thus cells bind to all spots in thearray in the same manner. In the optical method, cells bind to the arrayof spots of free amino groups by adhesion. Methods for attaching avariety of cell types to the same substrate for simultaneously bindingagainst these cell types also exist and can be used.

Nucleic Acid Arrays

Nucleic acid arrays are useful in a number of biological and clinicalstudies in which one or more genes are analyzed in parallel using thearray. Genetic disease is often caused by genes that are inappropriatelytranscribed—either too much or too little—or which are missingaltogether. Such defects are especially common in cancers, which canoccur when regulatory genes are deleted, inactivated, or becomeconstitutively active. Unlike some genetic diseases (e.g. cysticfibrosis) in which a single defective gene is always responsible,cancers that appear clinically similar can be genetically heterogeneous.For example, prostate cancer (prostatic adenocarcinoma) may be caused byseveral different, independent regulatory gene defects even in a singlepatient. In a group of prostate cancer patients, every one may have adifferent set of missing or damaged genes, with differing implicationsfor prognosis and treatment of the disease.

Comparative hybridization can serve two purposes in studying cancer: itcan pinpoint the transcription differences responsible for the changefrom normal to cancerous cells, and it can distinguish differentpatterns of abnormal transcription in heterogeneous cancers.Understanding the diverse basis of a cancer is crucial for inventingtherapies targeted to the different varieties of the disease, so thateach patient receives the most appropriate and effective treatment.

Cancers are common examples of genetically heterogeneous diseases, butthey are by no means the only ones. Diabetes, heart disease, andmultiple sclerosis are among the diseases for which genetic risk factorsare known to be heterogeneous.

Peptide-Nucleic Acids

In an alternative embodiment, peptide nucleic acids or oligomers, whichare analogs of nucleic acids in which, for example, the peptide-likebackbone is replaced with an uncharged backbone, can be used with thepresent invention. PNAs are well known in the art. References below giveextensive reviews of the use of these nucleic acid analogs in a widerange of applications, including surface and array-based hybridizationwherein PNAs are attached to surfaces and allowed to bind withsequence-complementary DNAs or RNAs.

For instance, oligomers of PNA can be used as the surface-attached probecomponents instead of DNA oligomers. A key advantage to using PNAs isthat the hybridization reaction with DNAs or RNAs, for example,(containing charged phosphate groups) is only weakly dependent (e.g.,the melting temperature) on ionic strength because there is much lesscharge repulsion as found with conventional DNA-DNA, etc. hybridization.Thus, one can use the surface-selective nonlinear optical technique tofollow a probe-target hybridization at any desired ionic strength. ThePNAs are commercially available (for instance via Applied Biosystems,Foster City, Calif.) or other analogs of DNA can be synthesized andused.

The following references are broad reviews of the use of PNAs:

-   Nielsen, et al. “Peptide nucleic acids-(PNA): Oligonucleotide    analogues with a polyamide backbone” Antisense Research and    Applications (1992) 363-372-   Nielsen, et al. “Peptide nucleic acids (PNAs): Potential Antisense    and Anti-gene Agents.” Anti-Cancer Drug Design 8 (1993) 53-63-   Buchardt, et al. “Peptide nucleic acids and their potential    applications in biotechnology” TIBTECH 11 (1993) 384-386-   Nielsen, P. E., Egholm, M. and Buchardt, 0. “Peptide Nucleic Acid    (PNA). A DNA mimic with a peptide backbone” Bioconjugate Chemistry    5 (1994) 3-7-   Nielsen “Peptide nucleic acid (PNA): A lead for gene therapeutic    drugs” Antisense Therapeutics 4 (1996) 76-84-   Nielsen, P. E. “DNA analogues with nonphosphodiester backbones” Annu    Rev. Biophys. Biomol. Struct. 24 (1995) 167-183-   Hyrup, B. and Nielsen, P. E. “Peptide Nucleic Acids (PNA):    Synthesis, Properties and Potential Applications” Bioorg. Med.    4 (1996) 5-23-   Mesmaeker, A. D., Altman, K.-H., Waldner, A. and Wendeborn, S.    “Backbone modifications in oligonucleotides and peptide nucleic acid    systems” Curr. Opin. Struct. Biol. 5 (1995) 343-355-   Noble, et al. “Impact on Biophysical Parameters on the Biological    Assessment of Peptide Nucleic Acids, Antisense Inhibitors of Gene    Expression” Drug. Develop. Res. 34 (1995) 184-195-   Dueholm, K. L. and Nielsen, P. E. “Chemistry, properties, and    applications of PNA (Peptide Nucleic Acid)” New J. Chem. 21 (1997)    19-31-   Knudsen and Nielsen “Application of Peptide Nucleic Acid in Cancer    Therapy” Anti-Cancer Drug 8 (1997) 113-118-   Nielsen, P. E. “Design of Sequence-Specific DNA-Binding Ligands”    Chem. Eur. J. 3 (1997) 505-508-   Corey “Peptide nucleic acids: expanding the scope of nucleic acid    recognition” TIBTECH 15 (1997) 224-229-   Nielsen, P. E. and Orum, H. “Peptide nucleic acid (PNA), a new    molecular tool.” In Molecular Biology Current Innovations and Future    Trends, Part 2. Horizon Scientific Press, (1995) 73-89-   Nielsen, P. E. and Haaima, G. “Peptide nucleic acid (PNA). A DNA    mimic with a pseudopeptide backbone” Chem. Soc. Rev. (1997) 73-78-   Ørum, H., Kessler, C. and Koch, T. “Peptide Nucleic Acid” Nucleic    Acid Amplification Technologies: Application to Disease    Diagnostics (1997) 29-48-   Wittung, P., Nielsen, P. and Norden, B. “Recognition of    double-stranded DNA by peptide nucleic acid” Nucleosid. Nucleotid.    16 (1997) 599-602-   Weisz, K. “Polyamides as artificial regulators of gene expression”    Angew. Chem. Int. Ed. Eng 36 (1997) 2592-2594-   Nielsen, P. E. “Structural and Biological Properties of Peptide    Nucleic Acid (Pna)” Pure & Applied Chemistry 70 (1998) 105-110-   Nielsen, P. E. “Sequence-specific recognition of double-stranded DNA    by peptide nucleic acids” Advances in DNA Sequence-Specific Agents    3 (1998) 267-278-   Nielsen “Antisense Properties of Peptide Nucleic Acid” Handbook of    Experimental Pharmacology 131 (1998) 545-560-   Nielsen “Peptide Nucleic Acids” Science and Medicine (1998) 48-55-   Uhlmann, E. “Peptide nucleic acids (PNA) and PNA-DNA chimeras: from    high binding affinity towards biological function” Biol Chem    379 (1998) 1045-52-   Wang “DNA biosensors based on peptide nucleic acid (PNA) recognition    layers. A review” Biosens Bioelectron 13 (1998) 757-62-   Uhlmann, E., Peyman, A., Breipohl, G. and Will, D. W. “PNA:    Synthetic polyamide nucleic acids with unusual binding properties”    Angewandte Chemie-International Edition 37 (1998) 2797-2823-   Nielsen, P. E. “Applications of peptide nucleic acids” Curr Opin    Biotechnol 10 (1999) 71-75-   Bakhtiar, R. “Peptide nucleic acids: deoxyribonucleic acid mimics    with a peptide backbone” Biochem. Educ. 26 (1998) 277-280-   Lazurkin, Y. S. “Stability and specificity of triplexes formed by    peptide nucleic acid with DNA” Mol. Biol. 33 (1999) 79-83-   Nielsen and Egholm “Peptide Nucleic Acids: Protocols and    Applications” (1999) 266 pp.-   Eldrup and Nielsen “Peptide nucleic acids: potential as antisense    and antigene drugs” Adv. Amino Acid Mimetics Peptidomimetics    2 (1999) 221-245-   Bentin, T. and Nielsen, P. E. “Triplexes involving PNA” Triple Helix    Form. Oligonucleotides (1999) 245-255-   Falkiewicz, B. “Peptide nucleic acids and their structural    modifications” Acta Biochim. Pol. 46 (1999) 509-529.

The following references are descriptions of the use of PNAs inarray-based detection, including means for attaching the PNA probes tothe solid surface.

-   Hoffmann, R., et al. “Low scale multiple array synthesis and DNA    hybridization of peptide nucleic acids” Pept. Proc. Am. Pept. Symp.,    15th (1999) 233-234-   Matysiak, S., Hauser, N. C., Wurtz, S, and Hoheisel, J. D. “Improved    solid supports and spacer/linker systems for the synthesis of    spatially addressable PNA-libraries” Nucleosides Nucleotides    18 (1999) 1289-1291.

ADDITIONAL EMBODIMENTS

The drawings illustrate various specific embodiments of an apparatus andsample using second or higher harmonic, sum or difference frequencygeneration.

FIG. 1 illustrates an embodiment wherein second harmonic light isgenerated by reflecting incident fundamental light from the surface.Light source 5 provides the fundamental light necessary to generatesecond harmonic light at the sample. Typically this will be a picosecondor femtosecond laser, either wavelength tunable or not tunable, andcommercially available. Light at the fundamental frequency (ω) exits thelaser and its polarization is selected using, for example a half-waveplate 10 appropriate to the frequency and intensity of the light (e.g.,available from Melles Griot, Oriel or Newport Corp.). The beam is thenfocused by lens 15 and passes through a pass filter 20 designed to passthe fundamental light but block the nonlinear light (e.g., secondharmonic). This filter is used to prevent back-reflection of the secondharmonic beam into the laser cavity which can cause disturbances in thelasing properties. The beam is reflected from a mirror 25 and impingesat a specific location and with a specific angle θ on the surface. Themirror 25 can be scanned if required using a galvanometer-controlledmirror scanner, a rotating polygonal mirror scanner, a Bragg diffractor,acousto-optic deflector, or other means known in the art to allowcontrol of a mirror's position. The sample surface 30 can be mounted onan x-y translation stage 35 (computer controlled) to select a specificlocation on the surface for generation of the second harmonic beam. Thesurface can be glass, plastic, silicon or any other solid surface thatreflects the fundamental or second harmonic beams. The sample surfacecan be enclosed and the surface in contact with liquid. Furthermore, thesample 30 can be fed or drained by microcapillary or otherliquid-transporting channels (not shown), pumps or electrophoreticelements, and these devices can be computer-controlled. The fundamentaland the second harmonic outgoing beams (at specific angles with respectto the surface, i.e., θ₁—they are typically nearly colinear indirection) then reflected from the surface and the fundamental isfiltered using a pass-filter 45 for the second harmonic beam, leavingonly the harmonic beam (2ω). The second harmonic is reflected frommirror 40, its polarization selected if necessary by polarizing optic50, and is focused using a lens 55 onto a detector 60. The lenses 15 and55 can also be any combination of lenses known in the art for focusingor beam shaping. If required, a monochromator 60 can also be used toselect a specific wavelength within the spectral band of the secondharmonic beam. The detector can be a photomultiplier tube, a CCD array,or any other detector device known in the art for high sensitivity. Forinstance, a photomultiplier tube operated in single-photon counting modecan be used. At the detector, the light generates a voltage proportionalto its intensity. Data is recorded for each location on the arraysurface as it is translated by the stage, scanned (or a combinationthereof) and an image is built up of the second harmonic intensitygenerated from each region on the surface.

Applicant envisions the use of sum or difference frequencies, where anapparatus set-up similar to FIG. 1 could be used, with the single lightsource 5 replaced by two light sources with two fundamental light beamsat frequencies ω₁, and ω₂. The sum or difference frequency (Ω) wouldthen be Ω=ω₁±ω₂. In the case where the sample surfaces are arrayscomprised of discrete elements, a single element or more than one inparallel can be addressed with the fundamental light. Furthermore,detection can be made on a single element or many in parallel dependingon the specific apparatus set-up.

FIG. 2 illustrates an embodiment in which total internal reflection(evanescent wave generation) is used to generate the second harmoniclight. Fundamental light (ω) is directed on to the surface of a prismelement 70. The beam is refracted at position (a) and passes through theprism, through an index matching film 75 and impinges on substrate 80.Prism 70 and substrate 80 are made of optically transparent materialsand are preferably of the same type. Prism 70 can be a Dove prism or anyother element which can support evanescent fields (e.g., waveguides,fibers and thin metallic films). The refractive index matching film 75can be an oil, but is preferably a compressible optical polymer such asthose disclosed by Sjodin, “Optical interface means”, PCT publication WO90/05317, 1990. The prism 75 and the substrate 80 can also be a unitary,integral piece made of the same material (i.e., without the indexmatching film). An evanescent wave is generated at the interface between80 and the medium in sample compartment 85 according to the indices ofrefraction in 80 and 85 and the angle of incidence of the beam at theirinterface. The electric field amplitude decays exponentially away fromthe substrate surface with a 1/e length ranging from nanometers tomicrons depending on several factors, including the surface electricpotential, the counterion density in the sample compartment (if any).The sample compartment can be filled with air, a gas, or a liquid suchas a solution or water. The ‘x’ marks on the surface of 80 facing thesample compartment emphasize that the sample of interest (e.g.,fabricated probes) are placed on this side. Substrate 80 can be a ‘chip’which can be slid out between 75 and 85, allowing for measurement ofdifferent substrates. Element 90 in the drawing refers to a port in thesample compartment for drawing liquid or gases in and out of thecompartment, for instance by pumps, electrostatic means, etc. The entiresample assembly can be mounted on an x-y translation stage 95 ifnecessary.

FIG. 3 illustrates an embodiment in which a slab-dielectric waveguide isused to deliver the fundamental light to the sample surface (the lightbeams are generated, directed and detected as in Drawing I with elements1-5 and 8-13). A parallel plate or dielectric waveguide can be used tocouple the fundamental light into a waveguide propagating mode. Thedrawing shows two slabs (110 and 115) and region (120). If the indicesof refraction of slab 115 and region 120 are less than the index ofrefraction of the light (for both fundamental and second harmonic), awaveguiding mode can be developed. This mode produces multiple internalreflections at the substrate which can be used to increase the amount ofsecond harmonic light generated by the interface. The fundamental beam100 can be coupled into the waveguide 110 using a diffraction grating105 scribed or embossed on the top surface of the waveguide, forexample. The fundamental is propagated along the length of the waveguideand makes multiple total internal reflections at the top and bottomsurfaces. The ‘x’ marks on substrate 110 denote the surface sample to bemeasured (i.e., containing the probes). If this interface generatessignificantly more second harmonic light than the interface betweenmaterials 110 and 115, the light intensity can be neglected. Forexample, if SH-labeled targets are bound to immobilized probes at the‘x’ locations and the atomic structure at the interface between 110 and115 is epitaxially matched, the interface 110/120 will generate muchmore second harmonic light than the interface 110/115.

In an alternative embodiment, a planar waveguide structure 110 is usedfor the solid substrate (FIG. 3). In this embodiment, a thin layer ofhigh index of refraction material 115 (the waveguide), such as TiO₂ orTa₂0₅, is deposited on top of the substrate 110 (typically glass). Athin diffraction grating 115 is scribed into this waveguide and lightfrom the laser 100 is coupled using this grating into the waveguide.Second harmonic light can be collected using lenses and filters anddetected with either a PMT-type device or a CCD camera.

FIGS. 4A-C illustrate an embodiment of a flow cell for carrying outprobe-target reactions. The flow cell is 3220 is shown in detail. FIG.4A is a front view, FIG. 4B is a cross sectional view, and FIG. 4C is aback view of the cavity. Referring to FIG. 4A, flow cell 3220 includes acavity 3235 on a surface 4202 thereon. The depth of the cavity, forexample, may be between about 10 and 1500 μm, but other depths may beused. Typically, the surface area of the cavity is greater than the sizeof the probe sample, which may be about 13×13 mm. Inlet port 4220 andoutlet port 4230 communicate with the cavity. In some embodiments, theports may have a diameter of about 300 to 400 μm and are coupled to arefrigerated circulating bath via tubes 4221 and 4231, respectively, forcontrolling temperature in the cavity. The refrigerated bath circulateswater at a specified temperature into and through the cavity.

A plurality of slots 4208 may be formed around the cavity to thermallyisolate it from the rest of the flow cell body. Because the thermal massof the flow cell is reduced, the temperature within the cavity is moreefficiently and accurately controlled.

In some embodiments, a panel 4205 having a substantially flat surfacedivides the cavity into two subcavities. Panel 4205, for example, may bea light absorptive glass such as an RG1000 nm long pass filter. The highabsorbance of the RG1000 glass across the visible spectrum (surfaceemissivity of RG1000 is not detectable at any wavelengths below 700 nm)substantially suppresses any background luminescence that may be excitedby the incident wavelength. The polished flat surface of thelight-absorbing glass also reduces scattering of incident light,lessening the burden of filtering stray light at the incidentwavelength. The glass also provides a durable medium for subdividing thecavity since it is relatively immune to corrosion in the high saltenvironment common in DNA hybridization experiments or other chemicalreactions.

Panel 4205 may be mounted to the flow cell by a plurality of screws,clips, RTV silicone cement, or other adhesives. Referring to FIG. 4B,subcavity 4260, which contains inlet port 4220 and outlet port 4230, issealed by panel 4205. Accordingly, water from the refrigerated bath isisolated from cavity 3235. This design provides separate cavities forconducting chemical reaction and controlling temperature. Since thecavity for controlling temperature is directly below the reactioncavity, the temperature parameter of the reaction is controlled moreeffectively.

Substrate 130 is mated to surface 4202 and seals cavity 3235.Preferably, the probe array on the substrate is contained in cavity 3235when the substrate is mated to the flow cell. In some embodiments, anO-ring 4480 or other sealing material may be provided to improve matingbet veen the substrate and flow cell. Optionally, edge 4206 of panel4205 is beveled to allow for the use of a larger seal cross section toimprove mating without increasing the volume of the cavity. In someinstances, it is desirable to maintain the cavity volume as small aspossible so as to control reaction parameters, such as temperature orconcentration of chemicals more accurately. In additional, waste may bereduced since smaller volume requires smaller amount of material toperform the experiment.

Referring back to FIG. 4A, a groove 4211 is optionally formed on surface4202. The groove, for example, may be about 2 mm deep and 2 mm wide. Inone embodiment, groove 4211 is covered by the substrate when it ismounted on surface 4202. The groove communicates with channel 4213 andvacuum fitting 4212 which is connected to a vacuum pump. The vacuum pumpcreates a vacuum in the groove that causes the substrate to adhere tosurface 4202. Optionally, one or more gaskets may be provided to improvethe sealing between the flow cell and substrate.

FIG. 4D illustrates an alternative technique for mating the substrate tothe flow cell. When mounted to the flow cell, a panel 4290 exerts aforce that is sufficient to immobilize substrate 130 located therebetween. Panel 4290, for example, may be mounted by a plurality ofscrews 4291, clips, clamps, pins, or other mounting devices. In someembodiments, panel 4290 includes an opening 4295 for exposing the sampleto the incident light. Opening 4295 may optionally be covered with aglass or other substantially transparent or translucent materials.Alternatively, panel 4290 may be composed of a substantially transparentor translucent material.

In reference to FIG. 4A, panel 4205 includes ports 4270 and 4280 thatcommunicate with subcavity 3235. A tube 4271 is connected to port 4270and a tube 4281 is connected to port 4280. Tubes 4271 and 4281 areinserted through tubes 4221 and 4231, respectively, by connectors 4222.Connectors 4222, for example, may be T-connectors, each having a seal4225 located at opening 4223. Seal 4225 prevents the water from therefrigerated bath from leaking out through the connector. It will beunderstood that other configurations, such as providing additional portssimilar to ports 4220 and 4230, may be employed.

Tubes 4271 and 4281 allow selected fluids to be introduced into orcirculated through the cavity. In some embodiments, tubes 4271 and 4281may be connected to a pump for circulating fluids through the cavity. Inone embodiment, tubes 4271 and 4281 are connected to an agitation systemthat agitates and circulates fluids through the cavity.

Referring to FIG. 4C, a groove 4215 is optionally formed on the surface4203 of the flow cell. The dimensions of groove, for example, may beabout 2 mm deep and 2 mm wide. According to one embodiment, surface 4203is mated to the translation stage. Groove 4211 is covered by thetranslation stage when the flow cell is mated thereto. Groove 4215communicates with channel 4217 and vacuum fitting 4216 which isconnected to a vacuum pump. The pump creates a vacuum in groove 4215 andcauses the surface 4203 to adhere to the translation stage. Optionally,additional grooves may be formed to increase the mating force.Alternatively, the flow cell may be mounted on the translation stage byscrews, clips, pins, various types of adhesives, or other fasteningtechniques.

In a further alternative embodiment, a suspension of beads, cells,liposomes or other objects comprise the probes (130) as shown in FIGS.5A-C. The scattered nonlinear light from such a sample—e.g., anisotropic sample in which each individual beads or other objects areabout a coherence length or farther apart—is generated in variousdirections with some distribution in intensity. Fundamental light istransmitted through the suspension (130) and the nonlinear radiationcollected. A number of modes of collecting the scattered nonlinear lightis available. For example, collection of the second harmonic can be inthe forward direction (A), at a right angle to the fundamental light(B), or using an integrating sphere approach (C). Part C shows anintegrating sphere 165 with the sample 150 placed inside. Fundamentallight (145) enters the entrance port (170), passes through the sample(150), undergoes a reflection at the sphere wall, and is stopped bybaffle (175). The scattered second harmonic light is collected from thesphere surface through exit port (155) and coupled out of the sphere bya fiber optic line (160). Cells are a convenient and natural system ofstudy for conformational changes in receptors, cell surface moleculesand other biological components. Beads can support phospholipid bilayers(e.g., with membrane proteins) or probes such as proteins or nucleicacids can be attached to their surface. The beads provide a large amountof distributed surface area in the sample and can be a usefulalternative to planar surface geometries, especially when thefundamental and nonlinear light is used in the transmission mode.

In an alternative embodiment (FIG. 6), the excitation light istransformed from a point-like shape into some other shape using variousoptics. For instance, the point-like beam shape of the fundamental beamcan be transformed into a line shape, useful for scanning the samplesurface. However, because the intensity of the nonlinear beam dependson, among other factors, the intensity of the fundamental (typically aquadratic dependence on the fundamental intensity), this transformationwill result in less nonlinear light intensity generated at a givenlocation. To generate a line-shape in the fundamental (which cantypically be a round point of ˜2 mm diameter), one can direct thefundamental beam into a microscope objective which has a magnificationpower of about 10 followed by a 150 mm achromat to collimate the beam aswell known in the prior art and as disclosed in detail in U.S. Pat. No.5,834,758. As shown in FIG. 6, the fundamental light 180 is a beam oftypically 2-3 mm diameter. This beam is directed through a microscopeobjective 185. The objective, which has a magnification power of 10,expands the beam to about 30 mm. The beam then passes through a lens190. The lens, which can be a 150 mm achromat, collimates the beam.Typically, the radial intensity of the expanded collimated beam has aGaussian profile. To minimize intensity variations in the beam, a mask195 can be inserted after lens 190 to mask the top and bottom of thebeam, thereby passing only the central portion of the beam. In oneembodiment, the mask passes a horizontal band that is about 7.5 mm.Thereafter, the beam passes through-a cylindrical lens 200 having ahorizontal cylinder axis, which can be a 100 mm f.l. made by MellesGriot. The cylindrical lens expands the beam spot vertically.Alternatively, a hyperbolic lens can be used to expand the beamvertically while resulting in a flattened radial intensity distribution.From the cylindrical lens, the light passes through a lens 205.Optionally, a planar mirror can be inserted after the cylindrical lensto reflect the excitation light toward lens 205. To achieve a beamheight of about 15 mm, the ratio of the focal lengths of the cylindricallens 200 and lens 205 is approximately 1:2, thus magnifying the beam toabout 15 mm. Lens 205, which in some embodiments is a 80 mm achromat,focuses the light to a line of about 15 mm.times.50 microns at thesample surface 210.

In an alternative embodiment shown in FIGS. 7A-B, probes are patternedin a two-dimensional array (A, top view of array on surface) where eachregion on the surface—{1,35} in this example—can be a differentoligonucleotide or protein sequence (or a combination of the same anddifferent sequences). Part B shows a side-view of the sample surface(220) in a well (215) containing the targets (225) shown here as proteinobjects with second-harmonic-active labels (X) attached. The well canhold liquid or buffer and serves to physically separate the contents ofthe well from other parts of the substrate or other elements in asubstrate array. The fundamental light can be multiplexed and eachresultant beam can be guided by individual mirrors to simultaneouslyscan different lines or regions within the array, thus increasing evenfurther the potential of the technique for high-throughput studies.

In an alternative embodiment, the method of Levicky et al. “UsingSelf-Assembly to Control the Structure of DNA Monolayers on Gold: ANeutron Reflectivity Study,” Journal of the American Chemical Society120: 9787-9792 (1998), or the method of Chrisey et al., “CovalentAttachment of Synthetic DNA to Self-Assembled Monolayer Films”, NucleicAcids Research 24, 3031, (1996), is used to attach the probe DNA to thesubstrate. In the method of Chrisey et al., as illustrated in FIG. 8, afused silica or oxidized silicon substrate is used (230) and derivatizedwith N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDA) (235). In oneembodiment, the EDA-modified surface is then treated with theheterobifunctional crosslinker (SMPB), whose succinimide ester moietyreacts with the primary amino group of EDA (240). A thiol-DNA oligomersubsequently (245) of base-pair sequence (xzzy) (where ‘xzzy’ representsthe entire sequence) reacts with the maleimide portion of the SMPBcrosslinker, to yield the covalently bound species shown (250).

In an alternative embodiment, elements in the surface array arephysically separated as illustrated in FIG. 9, allowing for differenttargets, target solutions, etc. to be added selectively to any or all ofthe elements. Part (A) is a top-view of the substrate (255) withpartitions or walls (260) separating the different well regions—in thisexample, 16 wells. Part (B) shows a side-view of a well (265) withattached probes (270). Such arrays are commonly found in the art, suchas the 96-well plates, etc. and are commercially available (FisherScientific, Inc. etc.)

In an alternative embodiment, a glass substrate surface can be coatedwith a layer of a reflective metal such as silver. The metallic layerwill increase nonlinear optical generation and collection. Biomoleculesor other particles can be attached to derivatized layers built on top ofthe metal. For instance, the metal can be coated with a layer of silicondioxide (SiO₂), then with a layer of aminosilane such as3-amino-octyl-trimethoxysilane. Oligonucleotides or polynucleotides canthen be attached to the aminosilane layer using linkers which connectthe 3′ or 5′ end of the oligo to the amine group. Alternatively, theoligos or polynucleotides can be adsorbed to the aminosilane layer. FIG.10 illustrates an embodiment of this type where a glass substrate (275)is derivatized with a Ag layer (280). A thin coat of SiO₂ is thendeposited on top of the silver layer (285) and derivatized with theaminosilane (290).

In a specific embodiment, nucleic acid or PNA microarrays can beobtained commercially or constructed according to public literature(e.g., http://cmgm.stanford.edu/pbrown/mguide/index.html). The surfacechemistry to be used is that found in Chrisey et al., “CovalentAttachment of Synthetic DNA to Self-Assembled Monolayer Films”, NucleicAcids Research 24, 3031, (1996), in which oligonucleotides are attachedto self-assembled monolayer silane films on fused silica slides.Silanization is accomplished viaN-(2-aminoethyl)-3-aminopropyltrimethoxysilane.

In other embodiments, oligonucleotides or PNAs can be attached to thesolid substrate via light-directed synthesis (S. A. Fodor, Science 277(1997), 393) or via chemical synthesis (e.g., Chrisey et al., “CovalentAttachment of Synthetic DNA to Self-Assembled Monolayer Films”, NucleicAcids Research 24, 3031, (1996)).

In still other embodiments, surfaces or microarrays microarrays ofoligonucleotides or PNAs can be obtained commercially or constructedaccording to public literature (e.g.,http://cmgm.stanford.edu/pbrown/mguide/index.html).

DNA microarrays can be obtained commercially or constructed, forexample, according to public literature (e.g.,http://cmgm.stanford.edu/pbrown/mguide/index.html). The surfacechemistry preferably to be used is that found in Chrisey et al.,“Covalent Attachment of Synthetic DNA to Self-Assembled MonolayerFilms”, Nucleic Acids Research 24, 3031, (1996), in whicholigonucleotides are attached to self-assembled monolayer silane filmson fused silica slides. Silanization is done viaN-(2-aminoethyl)-3-aminopropyltrimethoxysilane and Hoheisel, J. D.“Improved solid supports and spacer/linker systems for the synthesis ofspatially addressable PNA-libraries” Nucleosides Nucleotides 18 (1999)1289-1291 on glass or silica. The buffer or solution in contact with thePNA oligonucleotides can be chosen from a range of those known in theart. Hybridization and wash solutions are found in the art. For example,the web site: cmgm.stanford.edu/pbrown/protocols gives detailedinstructions for probe-target hybridization.

Microarrays can be mounted on an x-y translation stage and driven bypersonal computer (PC control) using a motorized translator (acquiredfrom Oriel, Inc.) or using one of the many procedures in the art (e.g.,V. G. Cheung et al., “Making and reading microarrays”, Nature Genetics(Suppl.), 1999, 21: 15-19).

In an alternative embodiment, bead-based fiber-optic arrays can be used(ref 34) in which light beams (e.g., fundamental and second harmonic)travel via total internal reflection along the path of the fiber. Thefundamental light is coupled into the bundle or individual opticalfibers and second harmonic light is generated at the tip surface andcollected back through the fiber. FIGS. 11A-B illustrate a fiber-opticbundle array. Part (A) shows a bundle of fiber optic cables (295) withwells at the distals ends for placement of beads (300). Part (B) shows aclose-up view of a single optical fiber. Fundamental light travels(.omega.) toward the distal end with the bead (305). Some fundamentallight is scattered back from the bead along with second harmonic light(2.omega.) and travels back through the fiber to the proximal end wherean optical train and detection system (not shown) separates thefundamental radiation from the second harmonic radiation. Bead (310) iscovered with probes.

In an alternative embodiment, green fluorescent protein (GFP) ornonlinear-active mutants thereof are used to label proteins in-vivo viamutagenesis according to procedures well known in the art. The GFP ormutants thereof are second-harmonic active and can serve as a built-inlabel of proteins. For example: a cell membrane receptor can be labeledwith GFP via mutagenesis. Cells containing this GFP-tagged receptorproduce some background second harmonic light when illuminated with afundamental beam in a surface-selective nonlinear optical technique.When ligands or other compounds which bind to the receptor and induceactivation—and a concomitant conformational change—the intensity and/orspectrum and/or time-course of the second harmonic light will change(due, for example, to a change in overall net orientation of the GFPprotein) as the GFP label moves slightly due to the conformationalchange on the receptor. The change in measured nonlinear opticalproperties are thus correlated with conformational change and ligandbinding.

In an alternative embodiment, the detector (65) of the nonlinearradiation in FIG. 1 is a photomultiplier tube operated in single-photoncounting mode. Photocurrent pulses can be voltage converted, amplified,subjected to discrimination using a Model SR445Fast Preamplifier andModel SR 400 Discriminator (supplied by Stanford Research Systems, Inc.)and then sent to a counter (Model 3615 Hex Scaler supplied by KineticSystems). Photon counter gating and galvo control through a DAC output(Model 3112, 12-Bit DAC supplied by Kinetic Systems) can be synchronizedusing a digital delay/pulse generator (Model DG535 supplied by StanfordResearch Systems, Inc.). Communication with a PC computer 29 can beaccomplished using a parallel register (Model PR-604 supplied by DSPTechnologies, Inc.), a CAMAC controller card (Model 6002, supplied byDSP Technologies, Inc.) and a PC adapter card (Model PC-004 supplied byDSP Technologies, Inc.).

In an alternative embodiment, a bandpass, notch, or color filter isplaced in either or all of the beam paths (e.g., fundamental, secondharmonic, etc.) allowing, for example, for a wider spectral bandwidth ormore light throughput.

In an alternative embodiment, an interference, notch-pass, bandpass,reflecting, or absorbant filter can be used in place of the filters inthe figures in order to either pass or block the fundamental ornonlinear optical beams.

According to another embodiment, detection of the nonlinear opticallight is achieved using a charge coupled detector (CCD) in place of aphotomultiplier tube or other photodetector. The CCD subsystemcommunicates with and is controlled by a data acquisition boardinstalled in a computer. Data acquisition board may be of the type thatis well known in the art such as a CIO-DAS 16/Jr manufactured byComputer Boards Inc. The data acquisition board and CCD subsystem, forexample, may operate in the following manner. The data acquisition boardcontrols the CCD integration period by sending a clock signal to the CCDsubsystem. In one embodiment, the CCD subsystem sets the CCD integrationperiod at 4096 clock periods. by changing the clock rate, the actualtime in which the CCD integrates data can be manipulated. During anintegration period, each photodiode accumulates a charge proportional tothe amount of light that reaches it. Upon termination of the integrationperiod, the charges are transferred to the CCD's shift registers and anew integration period commences. The shift registers store the chargesas voltages which represent the light pattern incident on the CCD array.The voltages are then transmitted at the clock rate to the dataacquisition board, where they are digitized and stored in the computer'smemory. In this manner, a strip of the sample is imaged during eachintegration period. Thereafter, a subsequent row is integrated until thesample is completely scanned.

In a specific embodiment, the nonlinear spectrum of a sample is measuredby measuring the nonlinear radiation (e.g., second harmonic radiation)at two or more spectral points or bands, using a monochromator, filteror other wavelength-selecting device to accomplish this.

In a specific embodiment, a monochromator (60) can be placed before thedetecting element in the device, in order to spectrally resolve thenonlinear optical radiation (FIG. 1).

In a specific embodiment, imaging techniques described in the art (Peleget al., “Nonlinear optical measurement of membrane potential aroundsingle molecules at selected cellular sites,” Proc. Natl. Acad. Sci. V.96, 1999, 6700-6704, or Campagnola et al., “High-resolution nonlinearoptical imaging of live cells by second harmonic generation,”Biophysical Journal 77 (6), 3341-3349 (1999), can be performed usingSHG-labeled components (such as labeled ligands or receptors) instead ofthe membrane intercalating dyes used in the art. These imagingtechniques can be used to image solid surfaces, cell surfaces or otherinterface using SHG-labeled components.

In a specific embodiment, channels (or microfluid) channels can be usedto introduce the components into the sample cell via positivedisplacement, pumping, electrophoretic means or other means known in theart for manipulating the flow of components into and out of a reactionchamber.

In a specific embodiment, the apparatus can be assembled into auser-closed product with a user-controlled interface (an LED panel, forexample, or PC-based software) with the option of inserting and removingdisposable substrates (e.g., biochips) with the attached probes.

In a specific embodiment, a photodiode, avalanche photodiode or otherphotoelectric detector (65) in FIG. 1 is used as the light detectionmeans.

In a specific embodiment, a surface array can be used that is in a fixedposition and the incident light beam scanned across the surface usingmethods well known in the art, such as a galvanometer mirror or apolygonal mirror.

An alternative embodiment comprises a scanning or imaging method where aphysical property of the nonlinear radiation is measured as function ofposition (x,y,z) in a sample. The scanning method can comprise acombination of both stage translation (x-y) and beam scanning, wherein,for example, the latter controls the incident position of thefundamental beam on the array surface.

In a specific embodiment, a stop-flow mixing chamber is used to rapidlymix the components in the sample cell.

In a specific embodiment, the proportionality constant (calibrationcurve of intensity of second harmonic light vs. concentration of targetsbound to attached probes) is determined by measuring the concentrationof targets using another method such as radiolabeling or fluorescencelabels of the targets. Once the calibration curve is known, for a givenprobe and target type (e.g., cDNA, RNA, size of oligos, etc.), theconcentration of bound target is determined using this relation and themeasured second harmonic intensity. This embodiment can be generalizedto any other nonlinear light beam emanating from the sample, includingthird harmonic, sum or difference frequency light.

In a specific embodiment, the nonlinear optical, surface-selectiveapparatus can comprise a unit without the light excitation source (e.g.,with sample compartment, filters, detectors, monochromator, computerinterface, software, or other parts) so that the user can supply his ownexcitation source and adapt its use to the methods described herein.

In an alternative embodiment, measurable information can be recorded inreal time.

Various Configurations of an Apparatus Using the Surface-SelectiveNonlinear Optical Technique in the Present Invention.

The apparatus for detection of the probe-target reactions or theireffects can assume a variety of configurations. In its most simple form,the apparatus will comprise the following:

-   -   i) a source of the fundamental light    -   ii) a detector for measuring the intensity of the second        harmonic or other nonlinear optical beams.

More elaborate versions of the apparatus will employ, for example: amonochromator (for wavelength selection), a pass-filter, color filter,interference or other spectral filter (for wavelength selection or toseparate the fundamental(s) from the higher harmonics), one or morepolarizing optics, a means of applying an electric field, one or moremirrors or lenses for directing and focusing the beams, computercontrol, software, etc.

The mode of delivering or generating the nonlinear optical light (e.g.,SHG) can be based on one or more of the following means: TIR (Totalinternal reflection), Fiber optics (with or without attached beads),Transmission (fundamental passes through the sample), Reflection(fundamental is reflected from the sample), scanning imaging (allows oneto scan a sample), confocal imaging or scanning, resonance cavity forpower build-up, multiple-pass set-up.

Measured information can take the form of a vector which can include oneor more of the following parameters: {intensity of light (typicallyconverted to a photovoltage by a PMT or photodiode), wavelength of light(determined with a monochromator and/or filters), time, substrateposition (for array samples, for instance, where different sub-samplesare encoded as function of substrate location and the fundamental isdirected to various (x,y) locations}. Two general configurations of theapparatus are: image scanning (imaging of a substrate-intensity,wavelength, etc. as a function of x,y coordinate) and spectroscopic(measurement of the intensity, wavelength, etc. for some planar surfaceor for a suspension of cells, liposomes or other particles).

The fundamental beam can be delivered to the sample in a variety ofways. FIGS. 12-16 are schematics of various modes of delivering thefundamental and generating second harmonic beams. It is understood thatin sum- or difference-frequency configurations, the fundamental beamswill be comprised of two or more beams, and will generate, at theinterfaces, the difference or sum frequency beams. For the purposes ofillustration, only the second harmonic generation case is described indetail herein. Furthermore, it shall be understood that the sample cell3 in all cases can be mounted on a translation stage (1-, 2-, or3-dimensional degrees of freedom) for selecting precise locations of theinterfacial interaction volume. The sample cell in all cases can befitted with flow ports and tubes which can serve to introduce (or flushout) components such as molecules, particles, cells, etc.

Transmission

FIG. 12A is a schematic of a configuration relying on transmission ofthe fundamental and second harmonic beams. The fundamental 320 (ω)passes through the sample cell 330 and interacts within a volume element(denoted by the circle) in which are contained one or more interfacescapable of generating the second harmonic beam 325 (2ω). The fundamentaland second harmonic beams are substantially co-linear as denoted by beam325. The sample cell can contain suspended beads, particles, liposomes,biological cells, etc. in some medium, providing interfacial areacapable of generating second harmonics in response to the fundamentalbeam. As shown, the second harmonic is detected co-linearly with thefundamental direction, but could alternatively be detected off-anglefrom the fundamental, for instance at 90° to the fundamental beam.

FIG. 12B is a schematic of another configuration relying on transmissionof the fundamental and second harmonic beams. The fundamental 335 isdirected onto a sample cell 345 and the second harmonic waves aregenerated at the top surface—this surface can be derivatized withimmobilized probes or with adsorbed particles, liposomes, cells, etc.The second harmonic waves 340 are generated within a volume elementdenoted by the circle at the interface between the top surface and themedium contained within cell.

FIG. 12C is a schematic of a configuration substantially similar to theone depicted in FIG. 2A except that the bottom surface of the samplecell 3, rather than the top, is used to generate the second harmonicwaves.

Total Internal Reflection

FIG. 13A is a schematic of a waveguide 4 capable of acting as a totalinternal reflection waveguide which refracts the fundamental 365 anddirects it to a location at the interface between the waveguide 380 anda sample cell 375. At this location, denoted by the circle, thefundamental will generate the second harmonic waves and undergo totalinternal reflection; the second harmonic beam will propagatesubstantially colinearly with the fundamental and exit the prism 380.Waveguide 380 will typically be in contact with air. In thisillustration, the waveguide 380 is a Dove prism.

FIG. 13B is a schematic of a configuration similar to the one depictedin FIG. 13A except that the waveguide 400 allows for multiple points oftotal internal reflection between the waveguide 4 and the sample cell395, increasing the amount of second harmonic light generated from thefundamental beam.

Fiber Optic

FIG. 14 depicts various configurations of a fiber optic means ofdelivering or collecting the fundamental or second harmonic beams. InFIG. 14A, the coupling element 410 between a source of the fundamentalwave and the fiber optic is depicted. The fundamental, thus coupled intothe fiber optic waveguide 405, proceeds to a sample cell 415. In FIG.14A, the tip of the fiber can serve as the interface of interest capableof generating second harmonic waves, or the tip can serve merely tointroduce the fundamental beam to the sample cell containing suspendedcells, particles, etc. In FIG. 14A, the second harmonic light iscollected back through the fiber optic.

FIG. 14B is identical to FIG. 14A except that a bead is attached to thetip of the fiber optic (according to means well known in the art). Thebead can serve to both improve collection efficiency of the secondharmonic light or be derivatized with probes or adsorbed species andpresenting an interface with the medium of sample cell 425 capable ofgenerating the second harmonic light.

FIG. 14C is identical to both FIGS. 14A and 14B except that collectionof the second harmonic light is effected using a solid-angle detector450.

Optical Resonance Cavity

An optical resonance cavity is defined between at least two reflectiveelements and has an intracavity light beam along an intracavity beampath. The optical cavity or resonator consists of two or more mirroredsurfaces arranged so that the incident light can be trapped bouncingback and forth between the mirrors. In this way, the light inside thecavity can be many orders of magnitude more intense than the incidentlight. This phenomenon is well known and has been exploited in variousways (see, for example, Yariv A. “Introduction to Optical Electronics”,2^(nd) Ed., Holt, Reinhart and Winston, N.Y. 1976, Chapter 8). Thesample cell can be present in the optical cavity or it can be outsidethe optical resonance cavity.

FIG. 15 is a schematic of an optical resonance power build-up cavityconfiguration. FIG. 15A is a schematic of an optical resonance cavity inwhich the sample cell 465 is positioned intracavity and the fundamentaland second harmonic beams are transmitted through it—a usefulconfiguration for sample cells containing suspended particles, cells,beads, etc. The fundamental beam 455 enters the optical resonance cavityat reflective optic 460 and builds up in power between reflectiveelements 460 and 462 (intracavity beam). Mirror 460 is preferably tilted(not perpendicular to the direction of the incident fundamental 455) toprevent direct reflection of the intracavity beam back into the lightsource. The natural reflectivity and transmissivity of 460 and 462 canbe adjusted so that the fundamental builds up to a convenient level ofpower within the cavity. The fundamental generates second harmonic lightin a volume element within the sample cell denoted by the circle.Reflective optic 460 can reflect the fundamental and the secondharmonic, while reflective optic 462 will substantially reflect thefundamental but allow the pass-through of the second harmonic beam 475which is subsequently detected. U.S. Pat. No. 5,432,610 (King et al.)describes a diode-pumped power build-up cavity for chemical sensing andit and the references it makes are hereby incorporated by referenceherein.

FIG. 15B is a schematic of an optical resonance power build-up cavityconfiguration in which the fundamental beam 475 enters the opticalcavity by reflection from optic 480. A second reflective optic element482 defines the optical resonance cavity. Element 490 is a waveguide(such as a prism) in contact with the sample cell 485 and allows totalinternal reflection of the fundamental beam at the interface between thewaveguide and sample cell surfaces, generating the second harmoniclight. Element 482 substantially reflects the fundamental beam butpasses through the second harmonic beam 495 which is subsequentlydetected.

Reflection

FIG. 16A is a schematic of a configuration involving reflection of thefundamental and second harmonic beams. A substrate 525 is coated with athin layer of a reflective material 520, such as a metal, and on top ofthis is deposited at layer 515 suitable for attachment of the probes oradsorption of particles, cells, etc. (e.g., SiO₂). This layer is incontact with the sample cell 510. The fundamental 500 passes through thesample cell 510 and generates a second harmonic wave at the interfacebetween layers 515 and 520. The fundamental and second harmonic waves505 are reflected back from the surface of layer 520.

FIG. 16B is substantially similar to FIG. 15A except that the secondharmonic and fundamental beams are reflected 535 from the interfacebetween the medium contained in sample cell 540 and layer 545. Layer 545is reflective or partly reflective layer deposited on substrate 550 andis suitable for adsorption of particles, cells, etc. or attachment ofprobes.

FIG. 16C is a schematic illustrating that only the sample cell 565 needbe used for a reflective geometry. The sample cell 565 is partly filledwith some medium 570 and the fundamental and second harmonic beams arereflected 560 from the gas-liquid or vapor-liquid interface at thesurface of 570.

Modes of Detection

Charge-coupled detectors (CCD) array detectors can be particularlyuseful when information is desired as a function of substrate location(x,y). CCDs comprise an array of pixels (i.e., photodiodes), each pixelof which can independently measuring light impinging on it. For a givenapparatus geometry, nonlinear light arising from a particular substratelocation (x,y) can be determined by measuring the intensity of nonlinearlight impinging on a CCD array location (Q,R) some distance from thesubstrate—this can be determined because of the coherent, collimated(and generally co-propagating with the fundamental) nonlinear opticalbeam) compared with the spontaneous, stochastic and multidirectionalnature of fluorescence emission. With a CCD array, one or more arrayelements {Q,R} in the detector will map to specific regions of asubstrate surface, allowing for easy determination of information as afunction of substrate location (x,y). Photodiode detector andphotomultiplier tubes (PMTS), avalanche photodiodes, phototransistors,vacuum photodiodes or other detectors known in the art for convertingincident light to an electrical signal (i.e., current, voltage, etc.)can also be used to detect light intensities. For CCD detector, the CCDcommunicates with and is controlled by a data acquisition boardinstalled in the apparatus computer. The data acquisition board can beof the type that is well known in the art such as a CIO-DAS 16/Jrmanufactured by Computer Boards Inc. The data acquisition board and CCDsubsystem, for example, can operate in the following manner. The dataacquisition board controls the CCD integration period by sending a clocksignal to the CCD subsystem. In one embodiment, the CCD subsystem setsthe CCD integration period at 4096 clock periods. By changing the clockrate, the actual time in which the CCD integrates data can bemanipulated. During an integration period, each photodiode accumulates acharge proportional to the amount of light that reaches it. Upontermination of the integration period, the charge is transferred to theCCD's shift registers and a new integration period commences. The shiftregisters store the charges as voltages which represent the lightpattern incident on the CCD array. The voltages are then transmitted atthe clock rate to the data acquisition board, where they are digitizedand stored in the computer's memory. In this manner, a strip of thesample is imaged during each integration period. Thereafter, asubsequent row is integrated until the sample is completely scanned.

Sample Substrates and Sample Cells

Sample substrates and cells can take a variety of forms drawing from,but not limited to, one or more of the following characteristics: fullysealed, sealed or unsealed and connected to flow cells and pumps,integrated substrates with a total internal reflection prism allowingfor evanescent generation of the nonlinear beam, integrated substrateswith a resonant cavity for fundamental power build-up, an optical set-upallowing for multiple passes of the fundamental for increased nonlinearresponse, sample cells containing suspended biological cells, particles,beads, etc.

Data Analysis

Data analysis operates on the vectors of information measured by thedetector. The information can be time-dependent and kinetic. It can bedependent on the concentration of one or more biological components,inhibitors, antagonists, agonists, drugs, small molecules, etc. whichcan be changed during a measurement or between measurements. It can alsobe dependent on wavelength, etc. In general, the intensity of nonlinearlight will be transformed into a concentration or amount of a particularstate (for example, the surface-associated concentration of a componentor the amount of opened or closed ion-channels in cell membranes) viathe detected change in nonlinear optical properties that are correlatedto conformational changes induced in the sample.

Details of the data analysis can vary from experiment to experiment.There is a large literature available for making correlations betweenconformation and fluorescence intensity (see the references by Glauneret al., Nature 402, 813 (1999), Ghanouni et al., Proc. Natl. Acad. Sci.,v. 98, 5997 (2001), and Ghanouni et al., Journal of BiologicalChemistry, v. 276, 24433 (2001), and references therein). Analogousprocedures are constructed for the nonlinear optical techniques. Forinstance, the square root of the intensity of second harmonic light(proportional to electric field amplitude of the light) is proportionalto the number of nonlinear-active species in a sample times theorientational average of the hyperpolarizability of the species. This isa well known relationship that can be used to quantify conformationalchange (and in turn binding affinity to a probe) with intensity of anonlinear beam. Kinetics and equilibrium properties of the reactions ofinterest can be determined via the measurements and appropriate dataanalysis.

Screening for Candidate Binding Partners

Candidate binding partners for binding a test molecule can be screenedthrough the detection of conformational changes on probe-target binding.The method of screening one or more candidate binding partners forbinding to a test molecule involves measuring the one or more physicalproperties of the one or nonlinear optical light beams emanating fromsaid sample comprising the test molecule and the one or more candidatebinding partners, where a change in the one or more physical propertiesof the nonlinear light beams relative to a value measured in the absenceof exposure to the one or more candidate binding partners is anindication of a binding event having occurred. In a preferred embodimentthe candidate binding partner is not attached to the surface.

The probes or targets of the present invention that can be used includebut are not limited to naturally occurring, artificially altered, orgenetically engineered, biological species or non-biological species.The candidates for probes or targets also include but are not limited toone or more of the following components: a nucleic acid, protein, smallmolecule, organic molecule, biological cell, virus, molecular beacon,liposome, receptor, antibody, agonist, antagonist, inhibitor, hapten,ligand, antigen, oocyte, homnone, protein, peptide, receptor, drug,lipid, ganglioside, enzyme, nucleotide, carbohydrate, cDNA,oligonucleotide, nucleoside, polynucleoside, polynucleotide, lipid,ganglioside, oligosaccharide, peptide nucleic acid (PNA), toxin, nucleicacid analog, ion channel receptor, G coupled-protein receptor. In aspecific embodiment, the probes can be patterned in an array format on asubstrate or solid surface, with the properties or chemical identity ofthe probes remaining constant or varying among regions of the array.

In one embodiment of the selection of candidate binding partners, anexternal electric field can be applied to the samples to create thenon-centrosymmetric condition required for the nonlinear opticaltechniques. For example, proteins (e.g., solubilized GPCRs) that arelabeled with a nonlinear-active label can be partially oriented in theelectric field. A background nonlinear light signal is measured. When aligand that binds to the protein is added to the medium, it triggers aconformational change in the protein (well known to those skilled in theart) that results in a change in the properties of the nonlinear lightsignal (e.g., a change in intensity). This scheme can be very useful inscreening libraries of ligands (small molecules, drugs, etc.) forbinding to proteins—i.e., high-throughput drug screening. In a preferredembodiment, the proteins are GPCRs, a well known as a class of proteinsthat are implicated in disease, and for which drugs can be developed.Drugs can be agonists, antagonists, inhibitors, etc. that interact(e.g., bind) with the receptors in some way. The following references(and references therein) describe different exemplary GPCRs that can beused:

-   P. Ghanouni et al., “Agonist-induced conformational changes in the    G-protein-coupling domain of the β₂ adrenergic receptor”, Proc.    Natl. Acad. Sci., v. 98, 5997 (2001).-   P. Ghanouni et al., “Functionally Different Agonists Induce Distinct    Conformations in the G Protein Coupling Domain of the β₂ Adrenergic    Receptor”, Journal of Biological Chemistry, v. 276, 24433 (2001).-   C. Bieri et al., “Micropatterned immobilization of a G    protein-coupled receptor and direct detection of G protein    activation”, Nature Biotechnology, 17, 1105 (1999).-   Helmreich, E. J. M. and Hofmann, K P, “Structure and Function of    Proteins in G-protein-coupled signal transfer”, Biochimica et    Biophysica Acta 1286, 285 (1996).-   Gether et al. (1995) J Biol Chem 270, 28628-28275, Fluorescent    labeling of purified β2 Adrenergic receptor.-   Turcatti et al. (1996) J Biol Chem 271, 19991-19998, Probing the    Structure and Function of the Tachykinin Neurokinin-Receptor through    Biosynthetic incorporation of fluorescent amino acids at specific    sites.-   Liu et al. (1996) Biochemistry 35, 11865-11873, Site-Directed    fluorescent labeling of P-glycoprotein on cysteine residues in the    nucleotide binding domains.-   Lodish, Harvey, et al., Molecular Cell Biology (4^(th) ed., 2000).

Screening for Modulators

The invention provides a method of screening one or more candidatemodulator molecules for the ability to modulate the interaction betweena test molecule and its binding partner. The action of modulator andinhibitor molecules have been previously described. Any change in theone or more physical properties of the nonlinear light beam emanatingfrom a sample comprising the test molecule, its binding partner and themodulator, relative to what was measured in the absence of exposure ofthe test molecule to the binding partners, serves as an indicator of theability of the candidate modulator molecules to modulate the interactionbetween the test molecule and its binding partner (i.e. to increase ordecrease their binding).

The invention can be used, for example, to monitor gene expression orfor studies involving drug screening or high-throughput screening wherea candidate drug is tested for binding, or its effect on probe-targetbinding, i.e., to reduce or enhance probe-target binding. In othercases, for example, a drug can be tested for efficacy by its ability tobind to a receptor or other molecule on the surface of a biologicalcell. In another specific embodiment, compounds that are potentialinhibitors of an agonist to a receptor are screened by testing forblocking of the agonist binding to the receptor, i.e., removal of aconformational change induced by the agonist when the receptor andagonist are also in the presence of an inhibitor candidate.

In a specific embodiment, the invention is used for drug screening orhigh-throughput screening where a candidate drug is tested for itsability to activate or inhibit a probe (e.g., a receptor, ion channelprotein, etc.). A drug candidate is tested for its ability to activate aconformational change in a probe-in this case, one seeks agonists of theprobe.

In an alternative embodiment, target-probe interactions can be measuredin the presence of some modulator of the interactions—the modulatorbeing, for example, a small molecule, drug, or other moiety, molecule orparticle which changes in some way the target-probe interactions (e.g.,has some affinity for the probe and blocks or inhibits target binding).The effect of a modulator on probe-target binding, where the target isknown to bind to the probes, is investigated using the nonlinear opticalmethod. The modulator can be added before, during or after the time inwhich the probe-target interactions occur.

In a specific embodiment, a biological probe-target binding reaction canbe measured in the presence of agonists, antagonists, drugs, or smallmolecules which can block, initiate or otherwise modulate the bindingstrength (e.g., equilibrium constant) of the said probe-target bindingreaction. This embodiment can be useful in many cases, for example whenone would like to know the efficacy of a drug's ability to block ormodulate a certain probe-target reaction for medical uses or basicresearch.

Detection of Conformational Chances

Conformational changes can be studied by the present invention at aninterface or in the bulk, where the conformational change leads to achange in one or more physical properties of one or more nonlinear lightbeams emanating from the sample. The conformational change can beinitiated by a biological or chemical binding event, or by a biologicalcomponent, drug, small molecule, agonist or antagonist binding to amolecule or a particle, and can be assayed as described herein forbinding interactions.

In a specific embodiment, a change in orientation or dipole momentoccurs in the interfacial region possessing a nonlinear susceptibilityas a result of some probe-target interaction. A nonlinear-active labelcan be attached to the probe of interest and binding of the probe tosome target (such as a drug candidate tested for its ability to activatethe probe and thereby induce a conformational change) in solutionresults in a change in orientation or dipole moment and this changes thenonlinear susceptibility of the interfacial region, and thus theproperties of the nonlinear beams (e.g., intensity, polarization,wavelength).

In another embodiment, a non-centrosymmetric region is created byapplication of an external field (EFISH technique). The region can beinterfacial, bulk or some combination thereof. Probes or targets (theprobes, targets or both are nonlinear-active, either intrinsically orlabeled using a nonlinear-active label) are poled by the electric fieldand this results in a background. When a binding reaction occurs betweenthe probes and targets, this can activate a conformational change in oneor both species or result in a change in dipole moment of species,resulting in a change in the measured nonlinear optical signals.

For use with nucleic acid hybridization (oligonucleotide,polynucleotide, RNA, etc.), target oligonucleotides can be exposed to asurface of an array on which are situated probe oligonucleotides. At theprobe oligonucleotide sequences in the array (corresponding to knownlocations) where sequence-complementary hybridization and anaccompanying conformational change occurs, the fundamental light wouldgive rise to a change in nonlinear optical signal, or a change in thebackground of such a signal. This can be detected and correlated withthe spatial location of the array element and hence the oligonucleotidesequence. For example, two major applications of nucleic acidmicroarrays are: 1) identification of sequence (gene or genemutation)—monitoring of DNA variations, for example; and 2)determination of expression level (abundance) of genes. There are manyformats that can be used for preparing the arrays. For example, in onecase probe cDNA (500˜5000 base pairs long) can be immobilized to a solidsurface such as glass using robot spotting and exposed to a set oftargets either separately or in a mixture (R. Ekins, F. W. Chu, Trendsin Biotechnology, 1999, 17, 217). Another format involves synthesizingoligonucleotides (20˜25 mer oligos) or peptide nucleic acids probesin-situ (on the solid substrate, Fodor et al., “Light-directed,Spatially Addressable Parallel Chemical Synthesis,” Science 251 (4995):767-773 Feb. 15, 1999) or by conventional synthesis followed by on-chipimmobilization. The array is then exposed to target DNA, hybridized, andthe identity or abundance of complementary sequences are determined.

Protein arrays can be prepared (see for example, G. MacBeath and S. L.Schreiber, “Printing Proteins as Microarrays for High-ThroughputFunction Determination”, Science 2000, 289, 1760-1763) to determinewhether a given target protein binds to the immobilized probe protein onthe surface. These arrays can be used to study small molecule binding tothe probe proteins.

Many reviews of microarray technology and applications are available inthe art. For instance, those of: Ramsay, “DNA chips-states-of-the-art,”Nature Biotechnology 1998, 16(1), 4044 (relevant portions of which areincorporated by reference herein); Marshall et al., “DNA chips-an arrayof possibilities,” Nature Biotechnology 1998, 16(1), 27-31 (relevantportions of which are incorporated by reference herein); S. A. Fodor,Science 277 (1997), 393 (relevant portions of which are incorporated byreference herein); D. H. Duggan et al., Nature Genet. 21 (Suppl.)(1999), 10 (relevant portions of which are incorporated by referenceherein); M. Schena et al., Science 270 (1995), 467 (relevant portions ofwhich are incorporated by reference herein); L. McAllister et al., Am.J. Hum. Genet. 61 (Suppl.) (1997), 1387 (relevant portions of which areincorporated by reference herein); and A. P. Blanchard et al., Biosens.And Bioelectron. 11 (1996) 687 (relevant portions of which areincorporated by reference herein).

The conformational changes also allow enable studying the degree orextent of binding between a probe and a target, utilizing the surfaceselective nonlinear optical techniques, through measuring theconformational effect the binding induces. In a specific embodiment,probes that are labeled with a nonlinear-active label are attached to asolid surface or substrate. When the probes bind to a target, theconformation of the probe changes, and the orientation of the label withrespect to the surface, and fundamental beam changes also. In apreferred embodiment the candidate binding partners (probes) are notattached to the surface. In another embodiment the nonlinear activelabel or moiety is attached to the target or probe molecules in vitro.The degree of probe-target binding can be correlated via the amount ofchange of a measured property of the nonlinear optical radiation (e.g.,intensity). For example, labeled oligonucleotides are attached to asolid substrate according to means well known in the art and exposed totargets to test for hybridization. When hybridization to a targetoccurs, the orientation of the label on the oligonucleotide changes.Microarrays known in the art (varying sequence of oligos in differentsurface locations) or substrates with uniform oligonucleotide sequencescan be used. Binding between surface-attached proteins and targets—otherproteins, ligands, etc. wherein the binding reaction triggers aconformational change are another exemplary embodiment with the presentinvention. In general, any surface-attached species that undergoes aconformational change when binding or interacting with any other speciesor stimulus can be studied with the present invention. Furthermore, thesurface-attached probes need not be labeled when using indicators whichare sensitive to minute changes in electric charge density or changesthereof. If the conformational change in a probe results in a change inthe arrangement of electric charges on a surface-even if this change istransient-indicators near the interface can be used to detect thesechanges and report on the conformational change.

In a specific embodiment, a MB analogue probe, described above, is usedto detect the degree or extent of binding. For instance, by labeling themolecular beacon probe with a nonlinear-active label and measuringwhether the label's orientation changes (via changes in nonlinearoptical intensity) at some interface, or in the bulk, one can studywhether target strands are complementary, and the extent to which theyare complementary since the amount of change that is measured innonlinear optical intensity can be correlated with the degree ofhybridization. In a specific embodiment, an Au nanoparticle is used toenhance the intensity of nonlinear optical radiation, such as secondharmonic generation scattered by an oxazole dye by several orders ofmagnitude when the nanoparticle and oxazole dye are in proximity to eachother. Upon hybridization of the probe to a complementary target, theintensity of the nonlinear optical radiation decreases and this decreasecan be quantitatively related to the amount of probe-targethybridization. The sensitivity of the technique is determined by, amongother factors, the background nonlinear optical signal beforehybridization occurs.

Variations on Uses of the Invention

Although the present invention can be used in many scientific areas ofanalysis and in particular, in the chemical and biological arts, thepresent invention can be especially useful in drug discovery or infundamental studies where compounds (targets) are tested for binding andability to activate probes, wherein the probes are ion channel proteins,GPCR proteins, or other receptors, or other molecules.

A wide flexibility is provided for the apparatus. Scanning, imaging,detection techniques at a fixed position, etc. can all be readily usedwith the present invention. Scanning of microarrays in the art includesconfocal-based schemes and non-confocal based schemes. U.S. Pat. No.5,834,758 (Trulson et al.—relevant portions of which are incorporated byreference herein) describes a non-confocal based scheme for imaging amicroarray using fluorescence detection. However, the sample should lievery flat in order to image only within a single focal plane for goodout-of-plane discrimination. Therefore, a very finely adjustabletranslation stage requiring specialized components is preferably be usedfor this purpose adding to the cost of the instrument and possibly thelifetime as well. The image quality of this type of apparatus can besensitive to mechanical vibrations. Furthermore, discrimination of theout-of-plane (non-surface bound) fluorophores places a limit on thesensitivity of the technique. U.S. Pat. No. 6,134,002 (Stimson etal.—relevant portions of which are incorporated by reference herein) isan example of a confocal scanning microscope device for imaging a sampleplane, i.e. a microarray. Although the confocal-based techniques havegood depth discrimination, the scan rate may be low due to descanningrequirements and the light throughput can be low, reducing the overallsignal to noise ratio and the sensitivity of the technique.

The invention can be used for studying binding processes between otherbiological components: cells with viruses; protein-protein interactions;protein-ligand; cell-ligand; protein-drugs, nucleic acid-drugs,cell-small molecule; cell-nucleic acid; peptide-cell, oligo orpolynucleotides, virus-cell, protein-small molecule, etc., and ingeneral, any binding reaction which results in a conformational change.Biomimetic membranes such as phospholipid supported bilayers (e.g., eggphosphatidylcholine) can also be used and are particularly useful whenstudies involve membrane protein probes.

Probes, targets, receptors, etc. can be rendered nonlinear-active (madeto possess a hyperpolarizability) by direct labeling or by using adecorator molecule or other candidate that has a binding affinity forthe probes or targets, and is itself intrinsically or renderednonlinear-active, and which will respond to a conformational change onthe probes or targets by shifting its position, by virtue of themolecular bond that binds the decorator to the probe or target. Anantibody to a receptor is an example of such a decorator molecule. Thedecorator molecule should not itself block the active region of thereceptor so that potential agonists, inhibitors, activators, etc. canbind to and produce action on the receptor.

Another example of the invention's use is to label receptors or othercomponents that have an affinity for some virus. When the virus binds orinteracts with the receptor or other component, this interaction willaffect the orientation of the label with respect to the direction of thefundamental beam, and thus change the properties of the measurednonlinear optical light (e.g., the intensity of the nonlinear light).

Other examples of the technique's use with arrays include cellulararrays, supported lipid bilayer arrays with or without membrane orattached proteins, etc. Many methods exist in the art for couplingbiomolecules (e.g., nucleic acid, protein and cells) to solid supportsin array format. A wide degree of flexibility may be used in providingthe means by which the arrays are created. They can involve, forexample, covalent or non-covalent coupling to the substrate directly, toa chemically derivatized substrate, to an intermediate layer of somekind (e.g., self-assembled monolayer, a hydrogel or other bio-compatiblelayer known in the art). The identity of the probes (e.g., proteinstructure or oligonucleotide sequence) can vary from site to site acrossthe solid surface, or the same probe can uniformly cover the surface.Targets can be of a single identity or a combination of targets withdifferent identities. The arrays can be prepared in a variety of waysincluding, but not limited to, ink jet printing, photolithography,micro-contact printing, or any other manner known to one skilled in theart of fabricating them.

Because the binding process can be measured in real time and in thepresence of bulk biological components due to the surface-selectivity ofthe nonlinear optical technique, equilibrium binding curves and kineticscan be measured, the bulk concentration of the components can be varied,and a “wash-away” step to remove unbound components, as is used withfluorescence-based detection, may be unnecessary.

A wide degree of flexibility is expected in the design of the apparatusincluding, but not limited to, the source of the fundamental light, theoptical train necessary to control, focus or direct the fundamental andnonlinear light beams, the design of the array, the detection system,and the use of a grating or filters and collection optics. The mode ofgeneration (irradiation) or collection can be varied including, forexample, the use of evanescent wave (total internal reflection), planarwave guide, reflection, or transmission geometries, fiber-optic,near-field illumination, confocal techniques or the use of a microcavityfor power build-up, or integrating detection system such as anintegrating sphere. A number of methods for scanning a microarray on asolid surface can be adapted for use. Examples include U.S. Pat. No.5,834,758 to Trulson et al. (1998), U.S. Pat. No. 6,025,601 to Trulsonet al. (2000), U.S. Pat. No. 5,631,734 Stem et al. (1997), and U.S. Pat.No. 6,084,991 to Sampas (2000)-relevant portions of which areincorporated by reference herein.

Because the second harmonic light beam makes a definite angle to thesurface plane, one can read-out the properties of the nonlinear opticalradiation, for instance, as a function of fundamental incidence positionin a two-dimensional array format, without needing to mechanicallytranslate the detector or sample and without extensive collectionoptics. In the ‘beam scanning’ embodiment, a mechanical translation ofsample surface or detector is not required-only a change in a directionand/or angle of the fundamental incidence on the sample (for a fixedsample and detector)—the apparatus offers much faster scanningcapability, improved ease of manufacturing and a longer lifetime.

When using the present invention to study an interface, the interfacecan comprise a silica, glass, silicon, silicon nitride, polystyrene,nylon, plastic, a metal, semiconductor or insulator surface, or anymixtures thereof, or any surface to which probes, such as biologicalcomponents, can adsorb or be attached. The interface can also includebiological cell and liposome surfaces. The attachment or immobilizationcan occur through a variety of techniques well known in the art. Forexample, oligonucleotides can be prepared via techniques described in“Microarray Biochip Technology”, M. Schena (Ed.), Eaton Publishing,1998-relevant portions of which are incorporated by reference herein.And, for example with proteins, the surface can be derivatized withaldehyde silanes for coupling to amines on surfaces of biomolecules (G.MacBeath and S. L. Schreiber, “Printing Proteins as Microarrays forHigh-Throughput Function Determination”, Science 2000, 289,1760-1763-relevant portions of which are incorporated by referenceherein). BSA-NHS (BSA-N-hydroxysuccinimide) surfaces can also be used byfirst attaching a molecular layer of BSA to the surface and thenactivating it with N,N′-disuccinimidyl carbonate. The activated lysine,aspartate or glutamate residues on the BSA react with surface amines onthe proteins.

The present invention can be applied to an ensemble of molecules or to asingle molecule—i.e., ensemble reaction measurements or asingle-molecule reaction measurement.

Supported phospholipid bilayers can also be used, with or withoutmembrane proteins or other membrane-associated components as, forexample, in Salafsky et al., Architecture and function of membraneproteins in planar supported bilayers: A study with photosyntheticreaction centers” Biochemistry 35 (47): 14773-14781 (1996) relevantportions of which are incorporated by reference herein, “Biomembranes”,Gennis, Springer-Verlag, Kalb et al., 1992 and Brian et al., “AllogeneicStimulation of Cyto-toxic T-cells by Supported Planar Membranes,”PNAS-Biological Sciences 81 (19): 6159-6163 (1984)-relevant portions ofwhich are incorporated herein. Supported phospholipid bilayers are wellknown in the art and there are numerous techniques available for theirfabrication, with or without associated membrane proteins. Thesesupported bilayers typically should be submerged in aqueous solution toprevent their destruction when they become exposed to air.

Probes can be part of a biological cell, liposome, bead, etc. thatnaturally form an interface capable of generating nonlinear opticalradiation due to their size (although they are nominallycentrosymmetric, their diameter is of the order of the wavelength oflight in the visible spectrum and this allows for generation of thenonlinear optical light according to well known art). Alternatively,probes can be molecules or particles (e.g., detergent-solubilizedreceptors) that are induced to orient by the application of an electricfield. The use of electric fields to create a non-centrosymmetric regioncapable of generating nonlinear radiation—e.g., second harmonic, sumfrequency or difference frequency generation, is well known in the priorart. One aspect of this prior art is often called ‘EFISH’-electricfield-induced second harmonic generation. In a specific embodiment, anapplied electric field is used to pole molecules within a region tocreate an ordered (non-centrosymmetric) region within a phase ormaterial; the resulting region is then capable of generating nonlinearoptical radiation.

In a specific embodiment, the probe-target hybridization can be measuredby detecting the intensity of nonlinear optical light (e.g., secondharmonic light) at some position on a substrate with surface-attachedprobes; the intensity of the second harmonic light changes as labeledtargets bind to the probes at the surface and become partially orientedbecause of the binding, thus satisfying the non-centrosymmetriccondition for generation of second harmonic light at the interface.Modeling of the intensity of light with concentration of probe-targetbinding complexes at the interface can be accomplished using a varietyof methods, for instance by calibrating the technique for a givenprobe-target interaction using radiolabels or fluorescence tags.Controls for non-specific binding of targets to the surface can beperformed according to procedures well known to one skilled in the art,for example: i) addition of deliberately non-complementary targets andmeasuring for surface-selective nonlinear optical signal, or ii) addingblockers which are known to prevent probe-target binding, addingcomplementary targets and measuring the resulting surface-selectivenonlinear optical signal. In case i) the surface-selective nonlinearoptical signal change upon addition of non-complementary targets will besubstantially lower than upon addition of complementary targets. In caseii) the signal change will be significantly lower in the presence ofblocker than in the absence of blocker.

EXAMPLES Example 1

A Molecular Beacon analogue (MB analogue) oligonucleotide, coupled to anonlinear-active dye, and purified, is purchased from a commercialsource such as Midland Certified Reagent Company (Midland, Tex.). Thenonlinear-active oxazole dye used is oxazole (SE)1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.)attached via an amine group at the 3′ end.

The oligonucleotide is placed into the sample well of an EFISH cell.There are a variety of EFISH cells available in the art. The sample celldescribed in the publication by C. G. Bethea (‘Experimental technique ofdc induced SHG in liquids: measurements of the nonlinearity of CH₂I₂″,Applied Optics 1975, 14, 1447) is used. The direction of the appliedelectric field is parallel to the electric field of the laser beam. Acommercial femtosecond mode-locked system (Mira 900 and Verdi 5W) isused as the fundamental source. The fundamental is directed into theregion of the cell between the electrodes. The back-reflected secondharmonic is blocked from entering the oscillator using a color filter;fundamental light beyond the sample is blocked using a color filter. Thesecond harmonic light is collected and focused onto a monochromator (CM110 CVI Laser) using plano-convex lenses (Melles Griot Inc.). The lightis detected using a Hamamatsu photomultiplier tube with a Bertan powersupply. The signals from the photomultiplier are sent to a StanfordResearch Systems SR400 photon counting unit and processed using a PC. Anelectric field is applied using a Bertan power supply and home-builtelectronics to pulse and synchronize the field with the laser pulses.

The dye-labeled MB analogue probes are poled by application of theelectric field when it is on. By comparing the average second harmonicintensity of the poled MB analogues in the absence and presence oftarget, the binding affinity (or sequence if it is unknown) of thetarget to the MB analogue can be measured. The SHG signal in the absenceof target represents a background measurement that is used as a‘baseline’ to compare with the signal in the presence of the target.

Upon addition of a perfect complementary target, the MB analogue probeis activated and this leads to a conformational change and therefore toa change in the average orientation of the nonlinear-active dye. Becausethe intensity of the measured SHG light is proportional to the averageorientation of the nonlinear-active dye, a change in dye orientationresults in a change in the intensity of the second harmonic beam.Targets with less than perfect complementarity to the MB analogue probesequence will bind with a lower affinity to the MB analogue probe andwill therefore activate a lower proportion of the MB analogue probemolecules and cause a smaller amount of the conformational change. Arelationship between binding affinity and the intensity of the secondharmonic beam can be readily developed according to procedures known inthe art that relate a change in nonlinear intensity to concentration ofthe nonlinear-active species.

Example 2

The β2 adrenergic receptor, a GPCR protein, is purified anddetergent-solubilized according to well known procedures (e.g., Ghanouniet al., 98(11): 5997 PNAS). The protein is labeled at an endogenouscysteine (Cys-265) with1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniumtrifluoromethanesulfonate (PyMPO epoxide, Molecular Probes), anonlinear-active dye, at 1:1 stoichiometry following standard proceduresand using the work of Ghanouni et al., 98(11): 5997 PNAS, as a guide.The nonlinear-active dye is attached to a part of the protein thatundergoes a conformational change when the protein is activated. Afterseparation of the non-covalently bound dye, the receptor is placed in amedium situated between two electrodes and through which passes afundamental beam (e.g., output of ˜1 W avg. Power, ˜150 fs pulses from aTi:Sapphire system such as the Verdi-Mira commercial system fromCoherent Inc.). The apparatus and sample preparation (e.g., applicationof electric field, electric field induced second harmonicgeneration-EFISH) are well known to one of ordinary skill in theart-standard optics are used to focus the fundamental on the sample andto collect the second harmonic radiation. FIG. 18 depicts the apparatusset-up and FIG. 17 depicts the sample holder with applied electricfield. An electric field is applied across the medium according to FIG.17, partially aligning the receptor and its bound dye and creating anon-centrosymmetric condition necessary for observation of SHG. Abackground signal intensity is measured at 400 nm using a color filter(BG-39, CVI Laser) to block the fundamental, a monochromator to selectwavelength (CM110, CVI Laser), a PMT (Hamamatsu) and single-photoncounting electronics (SR-400, Stanford Research Systems). The wavelength400 nm is selected to create a resonance enhancement effect via anelectronic transition of the dye. Ligand is added to the medium-a drugcandidate that is tested for binding, for example; if the ligand (drug)binds to the receptor, a conformational change in the probe (thereceptor) is induced which changes the nonlinear optical signalintensity. Known ligands (isoproterenol) and partial agonists(epinephrine, salbutamol and dobutamine) are used to calibrate thenonlinear optical response with amount of conformational change, i.e.,binding affinity.

In an alternate embodiment, the labeling reaction can be carried outusing various coupling chemistries and/or stoichiometries (label:probe)to determine which coupling chemistry gives the optimal signal in thenonlinear optical measurement. For instance, it may not be known apriori which sub-parts of the probes actually undergo a conformationalchange (positional shift) due to target activation and by undertaking avariety of labeling reactions (known in the art to be necessary to findoptimal labeling conditions in fluorescence labeling, etc.), it can bedetermined which chemistries lead to a label that undergoesconformational change when the probe is activated.

In an alternative embodiment, ion channels or receptors such as GPCRreceptors are labeled directly in biological cells with nonlinear-activelabels and/or enhancers. For instance, the publication by Glauner et al.(Nature, v. 402 813 (1999)) demonstrates fluorescent labeling of aShaker potassium ion channel in whole cells. A background signal ismeasured. When agonist binds to the receptors, it induces aconformational change in the receptors, changing the orientation of thelabels and thus the nonlinear optical signal (e.g., the signalintensity). A surface-selective nonlinear optical apparatus is used thusto measure ligand-gated conformational changes in the receptors via thelabels and/or enhancers in whole cells. Fundamental light is focusedonto a cell layer that has been cultured on Becton-Dickinson Falconplates, for instance—and the second or higher harmonic is collectedaccording to procedures well known in the art. In an alternativeembodiment, the labeled cells are suspended in a medium and fundamentallight is focused into a region of the sample containing these cells.FIG. 18 depicts an embodiment of this apparatus. Second harmonic lightis collected according to procedures known in the art, for instance at 0or 90 degrees with respect to the fundamental beam.

In an alternative embodiment, enhancers are suspended or dissolved inthe medium with labeled cells or molecules to enhance the nonlinearresponse of the cells or molecules.

In an alternative embodiment, probes are placed in artificial membranesor liposomes. If probes are expressed in cells, then can be purified andreconstituted into these membranes according to procedures well known inthe art.

In an alternative embodiment, the probes are in contact with, culturedon or patterned on surface that is itself in contact with a prism (or isthe underside of the prism).

The prism allows total internal reflection of the fundamental at theinterface containing the probes and thus high Fresnel factors (electricfield amplitudes) leading to higher nonlinear optical signals. In thismode of the set-up, the fundamental beam undergoes total internalreflection at the interface containing the probes and its evanescentwave is used to generate the nonlinear light. FIG. 2 illustrates anembodiment of this type. In FIG. 2, an index matching material or liquid(75) is used to couple the prism (70) to a substrate containing themicroarray (80) in contact with solution containing targets (85),whereby total internal reflection occurs at the interface betweenmaterial (80) and solution (85). The prism material can be, for example,BK7 type glass (Melles Griot) and the index matching material obtainedcommercially from Corning Corp. or Nye Corp.

In an alternative embodiment, the experimental set-up is as described inSalafsky and Eisenthal, “Protein adsorption at interfaces detected bysecond harmonic generation,” J. Phys. Chem. B, 104 (32): 7752-7755(2000), Salafsky and Eisenthal, “Second harmonic spectroscopy: detectionand orientation of molecules at a biomembrane interface,” Chem. Phys.Lett. 319 (5-6): 435-439 (2000), and references set forth therein. Afemtosecond pulsed laser (Mail-Tai, Spectra-Physics) is used as thesource of fundamental light at 800 nm operating at 80 MHz with <200 fspulses at 1 W average power. The laser beam directed onto the entranceaperture of a Dove prism (Melles Griot, BK-7) and focused with a concavelens (Oriel) (spot size ˜50 micron diameter). The Dove prism is mountedin a Teflon holder and in contact with buffer or distilled water. Thebeam undergoes total internal reflection (evanescent wave generation)within the prism and the fundamental and second harmonic beams emergeroughly collinearly from the exit aperture. A color filter is used toblock the fundamental light while passing the second harmonic to amonochromator (2 nm bandwidth slit). The monochromator is scanned from380-500 nm to detect the second harmonic spectrum. If necessary, thefundamental light wavelength can be tuned as well. A single photoncounting detector and photomultiplier tube are used to detect the outputof the monochromator and a PC with software are used to record the dataand control the monochromator wavelength. A background second harmonicsignal is measured before addition of ligand or other stimulus toproduce or test for conformational change in the probes.

Example 3

Oligodeoxyribonucleotides with suitable structures for molecular beaconsare selected and synthesized according to procedures known to one ofordinary skill in the art with a primary amine at the 3′ end and adisulfide group at the 5′ end and a biotin group that replaces a dT. Thefollowing MB analogue can be used, for example: 5′-CCT AGC TCT AAA TCGCTA TGG TCG CGC(Biotin dT)AG G-3′ (SEQ ID NO: 6). The amine-reactivenonlinear-active oxazole dye: oxazole (SE)1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.) isconjugated to the primary amine. In this coupling reaction, a 100 μlsolution containing 100 μM oligonucleotide dissolved in 0.1 M sodiumbicarbonate is reacted with 0.1 mg of the succinimidyl ester of the dyedissolved in 100 μl of dimethyl sulfoxide. The reaction mixture isstirred at room temperature for 2 hours. The reaction product ispurified with a Sephadex column (NAP-5; Amersham Pharmacia Biotech)equilibrated with 10 ml of 0.1 M triethylammonium acetate (pH 6.5).After purification, the 5′-end disulfide is cleaved and the freesulfhydryl is covalently attached to a 1.4 nm diameter gold cluster thatserves to enhance the nonlinear response of the oxazole dye (Nanogold;Nanoprobes), and which comes with one N-propylmaleimide and has beenpassivated with water-soluble phosphine ligands. The coupling of the Auparticle is achieved according to procedures well known to one ofordinary skill in the art. For example: the disulfide bond is cleavedwith dithiothreitol (DTT), and the oligonucleotide is purified of excessDTT before coupling to the gold. An amount of 10 μl of 1 M DTT is addedto 25 μl of oligonucleotide mixed with 75 μl of sodium bicarbonate, pH8.3. After a one hour incubation, the oligonucleotide solution ispurified using reverse-phase chromatography, as described. The fractionscontaining the activated oligonucleotide are purified using a Sephadexcolumn (NAP-5) equilibrated with water. Part of the elution product (37pmol to 370 pmol of DNA, suspended in 180 μl of water) is immediatelyreacted with 6 nmol of the monomaleimido-gold particles (Nanoprobes) inaqueous 20 mM NaH2PO4, 150 mM NaCl, 1 mM ethylenediamine tetraethylacetate (EDTA) buffer, pH 6.5, containing 10% isopropanol at 4° C. for24 h. Reaction products are analyzed by gel electrophoresis on a 10%nondenaturing acrylamide gel performed in Tris-borate EDTA (TBE) at 10V/cm.

The biotinylated MB analogue with attached nonlinear-active dye and goldnanoparticles are coupled to streptavidin-derivatized glass according towell known procedures. For example, the biotinylated MB analogues areimmobilized on the etched portion of a glass fiber. A batch of opticalfibers is used in a single immobilization cycle. About 2 cm of claddingis stripped away from the core by chemical etching at one end of thefiber probe. The fiber probe is perpendicularly dipped into a 49%hydrofluoric acid solution for 12 min. The HF solution is covered byheptane solvent. The etched fiber probe is washed with ultrapure waterbefore being used for the subsequent immobilization experiment.

Biotinylated MB analogues are immobilized on the etched portion of thefiber for DNA sensing. The etched fiber probes are first cleaned byimmersion in a 1:1 v/v concentrated HCl/MeOH mixture for 30 min., rinsedin water, and submerged in concentrated sulfurric acid for 30 min.Further rinsing and then boiling in water for 8-10 min follows.Silanization of the fibers is performed by immersing them in a freshlyprepared 1% (v/v) solution of DETA(Trimethoxysilylpropyldiethylenetriamine purchased from United ChemicalTechnologies, Bristol Pa.) in 1 mM acetic acid for 20 min at roomtemperature. The DETA-modified fiber probes are thoroughly rinsed withwater to remove excess DETA. The silanized fiber probes are dried undernitrogen and fixed by heating in a 120° C. oven for 5 min. Then thesilanized fibers are immersed in 0.5 mg/ml NHS-LC-biotin(sulfosuccinimidyl-6-(biotinamido) hexanoate purchased from Pierce,Rockford Ill.) in 0.1 M bicarbonate buffer (pH 8.5) for 3 hours at roomtemperature. Streptavidin (Sigma, St. Louis Mo.) is bound to the fibersurface by incubating the biotinylated fibers overnight at 4° C. in asolution containing 1.0 mg/ml of the streptavidin. Thestreptavidin-immobilized optical fibers are then immersed with abiotinylated MB analogue solution (10⁻⁶ M in 10 mM phosphate buffer atpH 7.0) for as long as 20 min or overnight at 4° C. to allow thebiotinylated MB analogue to be immobilized on the surface.

The optical fibers are interrogated optically using second harmonicgeneration by propagating a fundamental beam down the fiber andmeasuring the intensity of the second harmonic beam back-reflected afterit interacts evanescently with the MB analogue at the distal end of thefiber. In the absence of complementary targets, the oligonucleotideproduces a large amount of second harmonic light due to the proximity ofthe oxazole dye and the Au particle in the hairpin-loop structure of theMB analogue; the intensity of the second harmonic light in this case canserve as a background. In the presence of complementary targets, thehybridization reaction causes the dye and the Au particle to spatiallyseparate, greatly reducing the intensity of the second harmonic lightdetected. A linear relationship can be constructed between the amount ofhybridization that occurs and the intensity of the second harmonic lightand thus the derivatized optical fibers with detection system serves asan optical device for detection of complementary targets to the selectedprobe.

Example 4

Glass microspheres which are optically encoded with fluorescent dyes arederivatized with the MB analogues and an array of microspheres withdistinct oligonucleotide probes is prepared at the distal end of anoptical fiber as found in the art (e.g., U.S. Pat. No. 5,250,264, Waltet al.; U.S. Pat. No. 5,298,741 Walt et al.; U.S. Pat. No. 5,252,494Walt et al; U.S. Pat. No. 6,023,540 Walt et al.; U.S. Pat. No. 5,814,524Walt et al.; U.S. Pat. No. 5,244,813 Walt et al.; U.S. Pat. No.5,512,490 Walt et al. and commercially (Illumina Corporation, forexample). These arrays can be used to detect multiple target sequences.Alternatively, the optical encoding can be accomplished usingnonlinear-active dyes with different spectral characteristics from thebeacon-associated nonlinear-active dye so that the both the encoding andthe hybridization detection can be made using a nonlinear opticaltechnique such as second harmonic generation.

If the nonlinear MB analogues are used in homogeneous solution, anapplied electric field can be applied to the MB analogues in an EFISHmethod to create the required non-centrosymmetric condition.

In an alternative embodiment, one is interested in finding drugs,antagonists, agonists or other species which block or reduce the bindingof the MB analogues with targets—these compounds may be referred to as‘inhibitors’ or ‘blockers’. In this application, labeled targets arebound to probes at the interface. The inhibitors are added to thesample, and if the particular species being tested is successful inblocking or reducing the probe-target binding, the nonlinear opticallight measured will change—the background radiation in this embodimentis due to target-probe binding; the displacement of the targets from theprobes at the interface by the inhibitors leads to a change in thenonlinear optical light measured, for instance as a decrease inintensity of the nonlinear radiation generated by the interface or awavelength shift in the nonlinear radiation spectrum.

In an optional embodiment, controls to determine degree of non-specificnonlinear optical signals (e.g., not due to specific probe-targetbinding) can be performed according to standard procedures well known toone skilled in the art. In nucleic acid microarrays, for example, theintensity of the nonlinear optical signal at regions between theprobe-containing regions will produce a background signal that canincrease somewhat (but is substantially smaller than the signal due tospecific probe-target binding reactions) upon addition of targets thatare either complementary or non-complementary to the probes. Thisbackground signal can be accounted for by, for example, adding onlynon-complementary probes and measuring the nonlinear optical signal inregions containing probes and not containing probes. Measuring thenonlinear optical signals in the presence of blockers known to preventprobe-target binding is another control technique well known to oneskilled in the art for determining the amount of background orartifactual signal present in a larger signal of interest, in this casethe specific probe-target binding reaction.

In another embodiment of the invention, the amine-reactive oxazole dye(SE) 1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.) isreacted with a 1:1 molar ratio of ethylenediamine under the conditionsspecified by the Molecular Probes direction and is allowed to react tocompletion. The oxazole-based dye now contains a single amine group.This can be coupled to the primary amine on an oligonucleotide using ahomobifunctional crosslinking agent (Pierce, Rockford Ill.).

In an alternative embodiment, a nonlinear-active dye with attachedbiotin can be synthesized according to procedures known to one ofordinary skill in the art to create dye-biotin molecules that are thenonlinear-active analogues of the biotin-fluorescent dye moleculesuseful for immobilization of oligonucleotides.

Example 5

A peptide-nucleic analogue (PNA) molecular beacon (hairpin-loopstructure) of 18-25 base-pairs sequence is labeled with PyMPO, SE(Molecular Probes Corp.) at the 3′ or 5′ end (or, alternatively, atcytosine base-pair locations) according to procedures well establishedin the art. The PNA is placed in purified water solution and orientedwith an applied electric field to generate a non-centrosymmetric region.Addition of PNA molecules with complementary sequence to the labeled PNAwill cause a conformational change and thus a change in measuredproperties of the nonlinear optical beams.

PNAs (linear, non-hairpin loop) can also be attached to glass or silicasurfaces according to procedures well known to those skilled in the art.Addition of sequence-complementary DNA to purified water in contact withthe glass surface containing the PNAs results in a conformational changein the PNAs on the surface, and thus a change in the measured physicalproperties of the nonlinear optical light beam.

MISCELLANEOUS

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1-20. (canceled)
 21. A method for detecting a conformational change uponbinding of a test molecule to a candidate binding partner in a samplecomprising (a) contacting the test molecule with the candidate bindingpartner; (b) illuminating the contacted test molecule with a light beamat a fundamental frequency; (c) detecting the conformational change bymeasuring a physical property of a second-harmonic or sum-frequencylight beam emanating from the sample.
 22. The method of claim 21,further comprising selected a candidate binding partner based on thevalue of the physical property measured in step (c) relative to thecontrol value.
 23. The method of claim 21, wherein a change in the valueof the physical property measured in step (c) relative to a controlvalue indicates that the candidate binding partner modulates theconformation of the test molecule.
 24. The method of claim 21, whereinthe test molecule is contacted with the candidate binding partner at aninterface.
 25. The method of claim 24, wherein the interface is a lipidbilayer.
 26. The method of claim 21, wherein the candidate bindingpartner is attached to an interface.
 27. A method for detecting a shiftin average label angle of 5 degrees or less in a nonlinear-active labelupon binding of the test molecule to a candidate binding partner in asample comprising (a) contacting the test molecule with the candidatebinding partner; (b) illuminating the contacted test molecule with alight beam at a fundamental frequency; (c) detecting the shift bymeasuring a physical property of a second-harmonic or sum-frequencylight beam emanating from the sample.
 28. A method for screening acandidate conformation blocker of a test molecule comprising (a)contacting the test molecule with a known binding partner and acandidate conformation blocker, wherein the test molecule undergoes aconformational change upon the binding with the known binding partner;(b) illuminating the contacted test molecule with a light beam at afundamental frequency; (c) detecting the conformational change bymeasuring a physical property of a second-harmonic or sum-frequencylight beam emanating from the sample.
 29. The method of claim 28,wherein an absence of a change in the value of the physical propertymeasured in step (c) relative to a control value indicates that thecandidate binding partner blocks the conformation of said test molecule.30. The method of claim 28, further comprising selecting a candidateconformation blocker based on the value of the physical propertymeasured in step (c) relative to the control value.
 31. The method ofclaim 28, wherein the test molecule is contacted with the candidatebinding partner at an interface.
 32. The method of claim 31, wherein theinterface is a lipid bilayer.
 33. The method of claim 28, wherein thecandidate conformation blocker is attached to an interface.
 34. A methodfor screening different types of binding partners of a test molecule ina sample comprising (a) contacting the test molecule with the bindingpartners; (b) illuminating the contacted test molecule with a light beamat a fundamental frequency; (c) measuring a physical property of asecond-harmonic or sum-frequency light beam emanating from the sample;(d) determining the degree or extent of the binding between the testmolecule and the binding partners based on the value of the physicalproperty measured in step (c) thereby distinguishing different types ofbinding partners, wherein at least one of the binding partners induces aconformational change upon binding of a test molecule.
 35. The method ofclaim 34, further comprising selecting a candidate binding partner basedon the amount of change in the value of the physical property measuredin step (c) relative to a control value.
 36. The method of claim 34,wherein the amount of change in the value of the physical propertymeasured in step (c) relative to a control value indicates the degree orextent of the binding between the test molecule and the bindingpartners.
 37. The method of claim 34, wherein the binding partner is aninhibitor.
 38. The method of claim 34, wherein the test molecule iscontacted with the modulators at an interface.
 39. The method of claim38, wherein the interface is a lipid bilayer.
 40. The method of claim34, wherein the binding partners are attached to an interface.
 41. Themethod of claim 21, wherein the test molecule is a protein.
 42. Themethod of claim 21, wherein the candidate binding partner is a protein.43. The method of claim 21, wherein a nonlinear-active label is attachedto the test molecule, the binding partner, or the interface.
 44. Themethod of claim 21, wherein the conformational change is a localconformational change in the structure of a subpart of the test moleculethat occurs upon a binding event with the candidate binding partners.45. The method of claim 21, wherein the change in the physicalproperties of a second-harmonic or sum-frequency light beam is not dueto rotational motion of the test molecule.
 46. The method of claim 21,wherein the sample further comprises a modulator molecule.
 47. Themethod of claim 21, wherein the sample further comprises an enhancer.48. The method of claim 47, where the enhancer is attached to theinterface or the test molecule.
 49. The method of claim 47, where theenhancer is attached to the binding partner.
 50. The method of claim 21,wherein the control value is a value for the physical property measuredin the absence of exposure of the test molecule to the candidate bindingpartner.
 51. The method of claim 21, wherein the control value is avalue for the physical property measured upon binding with a controlbinding partner which is known to bind to the test molecule but not toproduce a conformational change.
 52. The method of claim 21, wherein thetest molecule undergoes a conformational change upon binding with thefirst binding partner.
 53. The method of claim 21, wherein the bindingpartner undergoes a conformational change upon binding with the firstbinding partner.
 54. The method of claim 21, wherein the test moleculeis labeled with a nonlinear-active label.
 55. The method of claim 54,wherein the nonlinear-active label is not native to the test molecule.56. The method of claim 21, wherein the binding partner is labeled witha nonlinear-active label.
 57. The method of claim 56, wherein thenonlinear-active label is not native to the binding partner.
 58. Themethod of claim 21, wherein the interface is labeled with anonlinear-active label.
 59. The method of claim 21, wherein thenonlinear-active label is a molecular beacon analogue.
 60. The method ofclaim 59, wherein the molecular beacon analogue comprises thenonlinear-active label and an enhancer.
 61. The method of claim 21,wherein the sample further comprises a decorator.
 62. The method ofclaim 61, wherein the decorator is attached to the nonlinear-activelabel.
 63. The method of claim 21, further comprising applying anexternal force field to the sample.
 64. The method of claim 63, whereinthe external force field is electrical field.
 65. The method of claim21, wherein the external force field is electrical field.